Biotechnology Advances 73 (2024) 108367

Contents lists available at ScienceDirect

Biotechnology Advances
journal homepage: [Link]/locate/biotechadv

Research review paper

Sustainable media feedstocks for cellular agriculture

Lutz Grossmann
Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA

A R T I C L E I N F O A B S T R A C T

Keywords: The global food system is shifting towards cellular agriculture, a second domestication marked by cultivating
Single cell protein microorganisms and tissues for sustainable food production. This involves tissue engineering, precision
Sustainable proteins fermentation, and microbial biomass fermentation to establish food value chains independent of traditional
Electrochemical ammonia
agriculture. However, these techniques rely on growth media sourced from agricultural, chemical (fossil fuels),
Green hydrogen
Second generation sugar
and mining supply chains, raising concerns about land use competition, emissions, and resource depletion.
Cellulose sugar Fermentable sugars, nitrogen, and phosphates are key ingredients derived from starch crops, energy-intensive
Gas fermentation fossil fuel based processes, and finite phosphorus resources, respectively. This review explores sustainable al­
Phosphate recovery ternatives to reduce land use and emissions associated with cellular agriculture media ingredients. Sustainable
alternatives to first generation sugars (lignocellulosic substrates, sidestreams, and gaseous feedstocks), sustain­
able nitrogen sources (sidestreams, green ammonia, biological nitrogen fixation), and efficient use of phosphates
are reviewed. Especially cellulosic sugars, gaseous chemoautotrophic feedstocks, green ammonia, and phosphate
recycling are the most promising technologies but economic constraints hinder large-scale adoption, necessi­
tating more efficient processes and cost reduction. Collaborative efforts are vital for a biotechnological future
grounded in sustainable feedstocks, mitigating competition with agricultural land and emissions.

1. Introduction Precision fermentation – i.e., the production of recombinant proteins –

has been utilized for foods since the 1980s, which resulted in the release
The food system is currently evolving towards a state that involves a of chymosin as the first FDA (USA) food-approved recombinant protein
widespread second domestication, which employs the large-scale culti­ in 1990 (Sutay Kocabaş et al., 2022). This technology has recently been
vation of microorganisms and tissues for food applications (RethinkX, expanded to other types of proteins such as β-lactoglobulin or ovalbumin
2019). This is the next step in the creation of a human-centric food (Aro et al., 2023; Hoppenreijs et al., 2024). Food products containing
system that started from the capture of animals (pre-domestication), these recombinant proteins have also been released to the market, for
followed by the first domestication period that involved the ‘cultivation’ example, by Bored Cow (New York, USA) or Remilk (Rehovot, Israel)
of macroorganisms (i.e., mainly animals). The use of microorganisms to (Wrobel, 2023). Lastly, microbial biomass fermentation involves the
transform foods has been used by humans for >10,000 years, but only production of cells (typically algae, fungi, and bacteria) for direct use in
within the 20th century more targeted approaches have been developed foods as a whole cell ingredient or as a raw material to extract certain
(Liu et al., 2018). Recently there has been increased interest in culti­ ingredients (lipids, proteins, pigments, etc.) (Buecker et al., 2022;
vating microorganisms and tissues to generate foods or food ingredients, Grossmann et al., 2019). These ingredients are sometimes also referred
which may allow for the production of more sustainable food products to as single-cell proteins. Quorn (Stokesley, UK), Solar Foods (Helsinki,
without the domestication of animals and plants (Eibl et al., 2021; Finland), and Nature's Fynd (Chicago, USA) are examples of companies
Grossmann and Weiss, 2021). Broadly these efforts can be summarized that use this approach.
under the term ‘cellular agriculture’, which consists of three main pil­ The main idea of these cellular agriculture cultivation techniques is
lars: (i) tissue engineering, (ii) precision fermentation, and (iii) microbial to establish food value chains that are less dependent on conventional
biomass fermentation. agriculture. However, all of them require growth media that rely on
Tissue engineering is used to obtain plant and animal tissue through agricultural, chemical, and mining supply chains. Especially tissue en­
in-vitro approaches. The most prominent tissue engineering endeavor is gineering requires sophisticated growth media that often rely on animal-
the production of cultivated meat (Ben-Arye and Levenberg, 2019). based ingredients such as fetal bovine serum that is obtained through

E-mail address: lkgrossmann@[Link].

[Link]
Received 11 February 2024; Received in revised form 11 April 2024; Accepted 23 April 2024
Available online 26 April 2024
0734-9750/© 2024 Elsevier Inc. All rights reserved.
 L. Grossmann
Table 1
Overview of technologies and existing challenges to obtain cellular agriculture media ingredients from sustainable feedstocks for biomass and precision fermentation. NR = not reported, NA = not applicable.
1st Generation Sugar Alternatives Nitrogen Phosphorous

Cellulosic Sugars Food & Agriculture Gaseous Chemoautotrophic Food & Agriculture Green Ammonia Biological Nitrogen Re-using Recycling
Sidestreams Sidestreams Fixation

Starting Material Lignocellulosic material from Carbohydrate-rich Water and electricity Protein-rich sidestreams Water, air, electricity Diazotrophic bacteria Spent media Phosphate-rich
forest, agriculture, industry, sidestreams materials (e.g., spent
aquatic, field trail sources media, wastestreams)
Technology Release fermentable sugars Release sugars from Green H2 production by Hydrolysis and Green H2 production by Intracellular Purify spent media Recover phosphates by
through pre-treatment and sidestreams or utilize electrolysis. H2 used directly or decomposition of electrolysis, which is used in(cyanobacteria) or to recover non- precipitation,
hydrolysis whole sidestreams for transformed into CH4 proteins into peptides, Haber-Bosch process extracellular (e.g., metabolized adsorption, filtration,
fermentation amino acids, and Azotobacter vinelandii) nutrients or biological uptake
ammonia nitrogen fixation
Feedstock Fermentable sugars (mainly Individual sugars or H2 or CH4 depending on the Peptides, amino acids, NH3 Intracellular = proteins/ Depends on Depends on method
Composition glucose, xylose, arabinose) whole sidestreams technology and ammonia peptides / extracellular purification
NH+ 4 method
2

1
Emissions 0.20–2.50 kg CO2 eq. per kg NR 1.2 kg CO2eq kg−SCP-Product (wind- NR 0.5 kg CO2 per kg green NH3 ( NR NR NA
sugar (Bello et al., 2021) energy based), 2.2 kg CO2eq kgSCP- Mayer et al., 2023)
− 1
Product (solar-based) (Järviö
et al., 2021; Sillman et al., 2019)
Challenges Consistent material availability, Fluctuating Availability and price of green Fluctuating composition, Scale up of green H2 Required production Potential Composition of
high capital ($0.02–1.10 kg− 1 composition, presence electricity, electrolyzer scalability presence of inhibitors, production, price reduction of volumes inhibitors present, recovered phosphates,
feedstock) and operating costs of inhibitors, continuous availability green H2 (needs to reach ≈ purification costs economics of recovery
($0.10–3.37 kg− 1 sugar) (Cheng continuous availability $1.9/kgH2 for $385.8/tonNH3)
et al., 2019) (Mayer et al., 2023)
Status Scaled for ethanol production ( Research Demonstration Research Demonstration Research Research Research/
de C. L. e Penalva Santos et al., Demonstrated
2023)

Biotechnology Advances 73 (2024) 108367

L. Grossmann Biotechnology Advances 73 (2024) 108367

Fig. 1. Simplified process flow diagrams for the production of glucose feedstock from starchy raw materials (first generation, e.g., wheat, corn) (Radeloff and Beck,
2014) and the production of second-generation fermentable sugar feedstocks from lignocellulosic materials. Capital expenditures (CapEx) and operational costs
(OpEx) taken from (Cheng et al., 2019). Icons from [Link].

ethically questionable methods (Lee et al., 2022a). The are currently fermentation.
tremendous efforts underway to replace such ingredients in tissue en­ This review discusses sustainable feedstocks and production pro­
gineering, which will not be discussed in this review because of the large cesses to obtain fermentable sugars, nitrogen compounds, and phos­
number of literature available regarding this topic (Hubalek et al., 2022; phates that can be deployed in biomass and precision fermentation
Lee et al., 2022a; Lee et al., 2023; O'Neill et al., 2021). media. The individual technologies and emissions as well as other
Biomass and precision fermentation media rely on three main in­ important considerations are summarized in Table 1. Using these
gredients that are derived from agricultural, chemical, and mining alternative ingredients will ultimately lower the land use and emissions
feedstocks: (i) fermentable sugars (usually monosaccharides such as associated with culture media formulation.
glucose and/or disaccharides such as sucrose), (ii) a nitrogen (ammo­
nium, urea, etc.), and (iii) a phosphorus source (i.e., phosphates). These 2. Sustainable alternatives to 1st generation sugars
ingredients are mixed at optimum ratios to obtain high growth rates
and/or maximum yield of the desired compound (Gao et al., 2007; Compounds derived from agriculture are typically the main
Wikandari et al., 2023) First, fermentable sugars are derived from starch biochemical energy sources for any biomass cultivation or precision
and sugar-containing crops such as wheat, corn, or sugarcane. The fermentation process. Glucose, sucroses, or other starch/sugar-derived
starch is hydrolyzed to glucose, which is then used to formulate the compounds are used to formulate the media. For example, mycopro­
media (Blanco et al., 2020; Kunamneni and Singh, 2005). This requires tein is often produced with malt extract (Pattillo, 2022) or glucose
agricultural land that could also be used for other purposes if such (Marsh, 1984), and glucose has also been used as an energy source for
fermentable sugars were derived from other types of raw materials. The microorganisms producing recombinant caseins (Pandya et al., 2021).
production of non-starch-derived fermentable sugars (2nd generation Thus, these processes ultimately rely on agricultural inputs from starchy
sugars) for generating bioethanol has received a lot of attention, but (corn, wheat, etc.) or sugary crops (sugarcane, sugar beet). To increase
alternative fermentable sugar feedstocks for food biotechnology have the independence of cultivation production processes from actual food
received only a little attention (Robak and Balcerek, 2020). Second, ingredients, three main approaches will be discussed: fermentable
nitrogen-containing media ingredients are typically a product of the sugars from lignocellulosic materials as well as sidestreams, and
Haber-Bosch process that currently runs on fossil fuels, which yields chemoautotrophic feedstocks.
ammonia utilizing natural gas as one of the main ingredients (Smith
et al., 2020). It is estimated that this process uses 1–2% of total global 2.1. Fermentable sugars for heterotrophs
energy, 3–5% of the globally produced natural gas, and is responsible for
1–3% of the total CO2 emissions (Kyriakou et al., 2020; Soloveichik, 2.1.1. Lignocellulosic substrates
2019; Song et al., 2023). Thus, sustainable alternatives are required to Glucose, sucrose, or other starch-derived compounds are ingredients
minimize the emissions of large-scale cellular agriculture production that can directly be consumed as a food or are derived from seeds and
systems. Lastly, phosphorous is a finite resource, which will be depleted plant material that can be consumed as a food. Coupled with starch
eventually. A comprehensive approach to re-use and recycle phosphates, shortages it is important to realize the dependence of current cultivation
therefore, needs to be implemented in biomass and precision approaches on food crops (Sharp, 2022). This dependence has been

3
L. Grossmann Biotechnology Advances 73 (2024) 108367

recognized for a few years by the scientific community and the food • chemical approaches like alkali, acid, oxidizing agents, organic sol­
industry (Bosque Foods, 2023; Jørgensen et al., 2007). One solution that vents, ionic liquids, and deep-eutectic solvents;
has been proposed is to release fermentable sugars from lignocellulosic • physicochemical techniques such as steam pretreatment with or
materials because of their abundant supply and low economic value. without a catalyst, wet-oxidation, and hydrothermolysis;
Lignocellulosic material is generally considered the plant biomass • biological methods that often involve digesting the material with
that consists mainly of the polymers cellulose, hemicelluloses, and lignin specific fungi.
but also contains smaller amounts of other compounds such as pectin,
protein, extractives, and ash (Jørgensen et al., 2007). It consists of 75% These methods are also often combined to achieve the desired out­
carbohydrates and is the most abundant renewable raw material glob­ comes as stated above. For example, the patented pretreatment method
ally (Cai et al., 2017). Due to these factors, it has been examined as a Sunburst™ uses a combination of twin-screw extrusion at high pressure
potential source to obtain fermentable sugars for cultivation processes (2.8 MPa) and high temperature (232 ◦ C) with diluted sulfuric acid and
that can replace glucose or other sugars derived from starchy or sugary steam injection as an effective pretreatment (Lumpkin, 2021). Typically,
food crops. In fact, this research dates back to the 1980s during which dilute-acid treatments and steam pretreatments are carried out to hy­
first experiments were conducted on the enzymatic conversion of Alfalfa drolyze the hemicelluloses, whereas alkali and organosolv treatments
and forage sorghums to release fermentable sugars (Linden et al., 1980). solubilize the lignin. Similar mechanisms have also been reported for
Since then many research endeavors have been carried out, which have biological treatments that can degrade lignin, hemicellulose, and cel­
been summarized in several reviews such as (Haldar et al., 2016; lulose through fungi fermentation. These methods are often combined
Jørgensen et al., 2007; Kumar and Sharma, 2017; Kumar et al., 2008; with milling and grinding operations that increase the porosity of the
Taha et al., 2016). The core ideas and challenges are summarized below material and the surface area (Galbe and Wallberg, 2019).
(Fig. 1). A few studies investigated pretreatment methods for single cell
Lignocellulosic materials are geographically evenly distributed, and protein production (Kumar et al., 2023; Tanaka and Matsuno, 1985;
it has been estimated that 10 to 50 billion tons are produced globally per Upcraft et al., 2021; Voutilainen, 2020; Voutilainen et al., 2021). In
year. Lignocellulosic biomass sources can be harvested from forests general, steam pretreatment is the most prevalent method, which has
(wood), agriculture (straw, stalks, sugarcane, etc.), industry (pine also been installed in demo plants to produce lignocellulosic ethanol
wood), water (seaweed), and field trails (miscanthus) (Usmani et al., (Galbe and Wallberg, 2019). The main considerations when choosing a
2021). Of these, wheat, rice, and sugarcane straw are abundant ligno­ pretreatment method are its effectiveness, the potential formation of
cellulosic materials with estimated worldwide production capacities per harmful compounds, and the economics behind the method. In a recent
year of 354, 731, and 181 million tons, respectively (Taha et al., 2016). study, Voutilainen (2020) compared steam explosion and ammonia
For the USA, barley straw, barley husks, canola straw, canola chaff, soaking as part of a mycoprotein production process from lignocellulosic
canola hulls, corn stover, rice straw, rice hulls, soy straw, soy hulls, feedstocks. The operating costs of the steam explosion were reported as
sugarcane trash, sugarcane bagasse, and wheat straw have been iden­ 477€ ($526) compared to 604€ ($666) for ammonia soaking. The au­
tified as promising lignocellulosic sources (GFI, 2023). Wheat straw, for thors, therefore, concluded that ammonia soaking has approximately
instance, consists of 32.9–50.0% cellulose, 24.0–35.5% hemicellulose, 25% higher production costs (based on straw as raw material) and was
and 8.9–17.3% lignin (Taha et al., 2016). Thus, there is a large potential considered in an in-depth techno-economic analysis (Voutilainen et al.,
to release fermentable sugars from these feedstocks. The release of these 2021). However, the authors also noted that due to acidic conditions in
sugars is, however, more challenging compared to sugars from edible the steam explosion process, inhibitors and toxic compounds such as
raw materials (so-called first generation sugars) in which the bio­ furfurals or acids like formic acid might be formed (EC, 2012).
polymers are often more readily accessible (Fig. 1). The cellulo­ Another study investigated the use of ionic liquids as a pretreatment
se–hemicellulose structures are stabilized by hydrogen bonds and lignin technique in a mycoprotein production process (Fusarium venenatum)
forms additional crosslinks with hemicellulose, which increases the that resulted in a glucose yield of 42.4% for food-grade ionic liquids,
resistance to chemical and biochemical degradation (Taha et al., 2016; which was lower compared to using non-food certified ionic liquids that
Zhang et al., 2015). Along with the high chemical diversity, this poses a resulted in a glucose yield of up to 92.8% (Upcraft et al., 2021). This
challenge because more sophisticated processing is necessary. A typical pretreatment process combined with the enzymatic breakdown of the
process to release fermentable sugars from lignocellulosic biomass lignocellulosic material resulted in mycoprotein production costs
typically involves two steps: pretreatment to increase the accessibility to ranging from $5.04 kg− 1 to $3.78 kg− 1 depending on the selected pro­
the biopolymers and the subsequent hydrolysis (‘saccharification’) cess parameters. The authors estimated that the final product price of
through chemical, enzymatic, or microbial processes (Kumar et al., the mycoprotein product would be around $21.80 kg− 1, which is more
2008). expensive than other currently available mycoprotein food products in
The aim of the pretreatment is to increase the accessibility of supermarkets. Thus, more research is required to obtain a better eco­
lignocellulose to the breakdown into its smaller constituents. Lignocel­ nomic understanding of the competitiveness of such a process that in­
lulose has a high ultrastructural, molecular, and chemical resistance due volves a relatively new pretreatment method.
to its partially crystalline and dense polymerized structure, which makes After the pretreatment, the material is subjected to the actual hy­
it inherently difficult to decompose (which is a natural defense strategy drolysis step to release the fermentable sugars. This involves the
of the plant against pests and weather) (Cheng et al., 2011; Houfani breakdown of cellulose and hemicellulose. Cellulose hydrolysis yields
et al., 2020). The pretreatment weakens the lignin and hemicellulose glucose, cellobiose, and cello-oligosaccharides, whereas hemicellulose
association, partially degrades the crystallinity of cellulose, removes hydrolysis results in different compounds depending on the composition
part of the lignin/hemicellulose, and increases the permeability of the of the raw material. For example, glucose, xylose, mannose, arabinose,
material (Kumar et al., 2009). Several pretreatment methods have been galactose, fucose as well as acetic, ferulic, glucuronic, and galacturonic
developed, which have been reviewed in several articles such as (Amin acids may be released from hemicellulose (Houfani et al., 2020). How­
et al., 2017; Galbe and Wallberg, 2019; Hendriks and Zeeman, 2009; ever, often xylose is the main fermentable sugar that is solubilized from
Kumar et al., 2009; Mankar et al., 2021). In brief and according to Galbe hemicellulose followed by arabinose and other sugars/sugar acids,
and Wallberg (2019), pretreatments can be loosely classified based on especially for straw-based feedstocks (which has often been studied for
the employed principle (Fig. 1): single-cell protein cultivation) (Parsin and Kaltschmitt, 2024; Rodrí­
guez-Sanz et al., 2022; Silva et al., 2013) (Table 1). Thus, the organism
• physical processes such as milling, grinding, extrusion, irradiation, that is cultivated on fermentable sugars coming from hemicellulose-
and sonication; containing raw materials needs to be able to utilize these non-glucose

4
L. Grossmann Biotechnology Advances 73 (2024) 108367

sugars for their energy metabolism. been used for 2nd generation ethanol production but a few studies also
The hydrolysis step to transform lignocellulose into fermentable produced fermentable sugars for single-cell protein production (Kas­
sugars can be carried out by chemical or enzymatic methods (Fig. 1). perski, 2023; Upcraft et al., 2021; Zhang et al., 2023). For example,
Chemical hydrolysis involves the treatment of the pretreated lignocel­ Kasperski (2023) released glucose from sugarcane bagasse to grow
lulosic biomass with acids like sulfuric acid, acetic acid, phosphoric acid, different bacterial and fungal strains. The bagasse was pretreated using
hydrochloric acid, and nitric acid (Haldar et al., 2016). The acid hy­ steam explosion at 185 ◦ C for 10 min, which resulted in a composition of
drolysis can be performed either under concentrated or diluted condi­ 36.5% cellulose, 18.7% hemicellulose, and 24.4% lignin. The authors
tions. In the concentrated regime, the material is mixed with reported a conversion of around 80% of the cellulose into glucose using
hydrochloric or sulfuric acid at temperatures of up to 100 ◦ C and con­ the Cellic® CTec3 enzyme product at a solid load of 15% (w/v) and an
centrations typically above >30%, which can result in glucose yields of enzyme concentration of 7.5 FPU/g DW. This yielded 42 g/L of glucose
>90% (AAF, 1980; Chen, 2015; Clausen and Gaddy, 1993; Clausen and for the fermentation process. It has to be noted that the authors only
St, 1993; Kong-Win Chang et al., 2018). In the diluted approach, similar reported the concentration of glucose and other sugars were not re­
acids such as sulfuric or hydrochloric acids are used, but at lower con­ ported even though the employed enzyme mixture also has hemi­
centrations that are commonly below 10% (often at a maximum of up to cellulase activities (Ilić et al., 2023). However, Bacillus subtilis CAB1111,
3–4%) (Chen, 2015; Lenihan et al., 2010; Tsoutsos, 2010). To achieve a Lactobacillus delbrueckii, Streptococcus thermophilus, Fusarium venenatum,
successful hydrolysis, the temperature is raised to around 100–240 ◦ C Pleurotus ostreatus CAB13, and Saccharomyces cerevisiae CAB79 were
for several minutes. Both processes operate under very harsh conditions cultivated separately using the released glucose feedstock. Especially
due to the corrosive nature of the employed acids, which also results in S. cerevisiae tolerated the inhibitors that formed during the pretreatment
the potential formation of inhibitors that can affect the subsequent (mainly acetic acid) and metabolized the present glucose completely
fermentation (Houfani et al., 2020). Thus, there have been substantial with a final biomass concentration of 20.4 g/L. This was in contrast to
efforts over the last decades to develop less harsh hydrolysis methods the filamentous fungi Fusarium venenatum and Pleurotus ostreatus which
using enzymes to break down lignocellulosic materials (Teter, 2012). were not able to grow in the sugar solution obtained from the bagasse.
During enzymatical hydrolysis, the lignocellulosic material is hy­ This highlights the need to either choose the right organism that can
drolyzed with mixtures of cellulases and hemicellulases (Østby et al., tolerate the formed inhibitors or to remove the inhibitors before the
2020). At least three enzymes are needed to break down the cellulose cultivation process.
part of the material: endoglucanases (cleaves internal β-1,4 glycosidic To overcome inhibition of the microorganism by the compounds
bonds), cellobiosidase (exoglucanases that release cellobiose from the formed during the pretreatment, Zhang et al. (2023) detoxified
chain end), and β-glucosidases (mainly hydrolyzes the cellobiose to fermentable sugars derived from wheat straw (obtained through dilute
release glucose) (Berlemont and Gerday, 2011). This is in contrast to the acid pretreatment and Cellic CTec2.0 enzyme hydrolysis) using Paeci­
breakdown of hemicellulose, which requires a set of different enzymes lomyces variotii FN89 to degrade acetic acid, furfural, and 5-hydroxyme­
because of the complex heterogeneous composition that also differs thylfurfural. High inoculum concentrations and short timeframes were
between different raw materials (Østby et al., 2020). In fact, some of the employed to not let the detoxifying organism consume too much glucose
developed enzyme cocktails may contain up to 80 different enzymes to before the actual cultivation. The detoxified solution contained 120 g/L
effectively release the fermentable sugars (Van Dyk and Pletschke, of fermentable sugars of which were 80 g/L glucose and 40 g/L xylose.
2012). According to Ferreira et al. (2021) the following enzyme mixture These sugars were completely metabolized by Trichosporon cutaneum
with its specific functions is a good starting point to release fermentable MP11 within >36 h and the protein content reached 24.4 g/L. Sur­
sugars from hemicellulose: prisingly, the lignocellulose-derived sugars outperformed a standard
medium in terms of protein productivity and protein content in the
• endo-1,4-β-xylanases EC [Link] (hydrolyzes internal xylan chains), obtained biomass. The study concluded that from 4.8 t of wheat straw, 1
• exo-β-1,4-xylanases (cleaves reducing ends of xylan resulting in t of microbial biomass with a protein content of 29.4% can be produced.
xylose and xylobiose), In conclusion, lignocellulosic materials are a promising feedstock to
• 1,4-β-xylosidases EC [Link] (acts at nonreducing ends of xylan), produce fermentable sugars for bioproduction processes to obtain mi­
• α-l-arabinofuranosidase EC [Link] (releases arabinose units from crobial biomass or other compounds. Several challenges that are con­
the xylan's sidechain), nected to the pretreatment and hydrolysis operations have been
• α-glucuronidase EC [Link] (cleaves glucuronic acid groups), identified over the years and will be discussed below.
• acetyl xylan esterase EC [Link] (facilitates deacetylation of xylan),
and [Link]. Challenges. Hydrolyzing lignocellulosic materials to release
• feruloyl/coumaroyl esterases EC [Link] (breaks down ester bonds fermentable sugars has been identified as one possible solution to use
between the lignin's phenolic compounds and hemicellulose) as well less food crops for fermentation processes. There have been extensive
as mannan cleaving enzymes for hemicellulose types that contain global efforts to establish lignocellulosic supply chains for the produc­
substantial amounts of mannan (Varnai, 2012). tion of ethanol through fermentation. The technique itself dates back to
1819 but major efforts were fueled by the oil embargo by Arab countries
In addition to these main enzymes, oxidizing and auxiliary enzymes in the 1970s, which jumpstarted the interest in using other energy
such as laccases and pectinases are needed to decompose the lignin part sources than oil for transportation (Smith, 2019). Initially, food crops
and other components of the biomass (Taha et al., 2016; Varnai, 2012). were investigated as feedstocks for ethanol production but the ‘fuel vs.
Several commercial enzyme mixtures have been released from Novo­ food’ debate shifted the focus to lignocellulosic materials in later de­
zymes for this purpose (Bagsværd, Denmark). These include Celluclast® cades. However, even though large-scale research and development
1.5 L (Endo-1,4-β-d-glucanase), Viscozyme® L (Endo-1,4-β-xylanase, projects have been carried out since the beginning of this millennium,
non-reducing end α-L-arabinofuranosidase, endo-1,4-β-d-glucanase), cellulosic ethanol has not been a major success due to several technical
Viscoferm® (Endo-1,4-β-xylanase, endo-1,3-(1,4)-β-d-glucanase), and challenges (Fialka, 2018; Kramer, 2022). In fact, only a few commercial
Cellic® CTec3 HS (cellulase and hemicellulase mixture). But also other cellulosic ethanol production plants are operational (Padella et al.,
companies such as Genencor (Palo Alto, CA, USA) and their Accelerase 2019). Some of these challenges may also be applicable to microbial
product series have been developed to hydrolyze lignocellulosic biomass biomass production or precision fermentation when fermentable sugars
(Ilić et al., 2023; Østby et al., 2020; Prévot et al., 2013; Radenkovs et al., are used that are derived from lignocellulosic materials.
2021; Yang et al., 2017). Technically it is possible to transform most of the polymeric sugars in
These commercial enzymes and enzyme mixtures have primarily

5
L. Grossmann Biotechnology Advances 73 (2024) 108367

lignocellulosic biomass into fermentable sugars (Beckendorff et al., an enzyme concentration of 10 mg cellulase g− 1 cellulose and a 20%
2023; Chundawat et al., 2017; Liu et al., 2021; Nielsen et al., 2017; Xu solids concentration (Himmel, 2019). Other strategies to reduce the
et al., 2010; Yu et al., 2020), even though efforts to increase the glucan- costs of lignocellulosic biomass conversion are (DoE, 2020):
to-glucose and xylan-to-xylose yield at low enzyme concentrations are
still active fields of research (Himmel, 2019). The main challenge in • Employing efficient conversion methods to fully utilize all compo­
converting lignocellulose into fermentable sugars is, however, the nents within the feedstock;
associated costs of the process. Converting starch into glucose is a • Decreasing capital expenditures and facility operating costs through
relatively straightforward process compared to the required treatment improved process integration or the adoption of novel processing
of the lignocellulosic material. Capital costs are considerably higher for routes;
building a lignocellulosic conversion plant that is used for ethanol • Leveraging existing infrastructure for enhanced operational
production compared to corn-based ones (Peplow, 2014). This is related efficiency;
to the recalcitrance of the lignocellulose matrix compared to starch, • Decreasing feedstock expenses by incorporating waste and low-
which is much more readily accessible. As described before, pretreat­ quality feedstocks; and
ment and complex enzyme mixtures are needed to transform the cellu­ • Crafting high-value products through strategic development
lose and hemicellulose into fermentable sugars. It has been estimated initiatives.
that the required enzyme concentration has to be onefold higher to
hydrolyze lignocellulose material compared to starchy biomass (Balan, As mentioned before, these analyses have mainly been carried out for
2014). In fact, on average the operational costs of a lignocellulose transforming lignocellulose into bioenergy, but not many studies exist
conversion plant were reported to be 42% of the total costs, with the that looked into the value chain of transforming lignocellulose into
remaining being the feedstock costs (33%) and capital costs (25%) fermentable sugars for precision and biomass fermentation. One study
(Witcover and Williams, 2020). In detail, it has been recently reported evaluated the costs of precision and biomass fermentation using
that first-generation crops (i.e., starchy food crops such as corn) are fermentable sugars derived from wheat straw (Voutilainen et al., 2021).
associated with lower capital costs (facilities, equipment, and con­ The authors modeled a process based on treating the straw by steam
struction costs) that range from $0.01 to $0.13 kg− 1 feedstock and explosion followed by enzymatic hydrolysis that resulted in sugar costs
production costs that range from $0.22 to $0.55 kg− 1 sugar. This is in of 622 € ($677)/ton of sugar (which is higher compared to regular su­
contrast to cellulosic sugar production which is associated with higher crose which was 210 € ($229) to 310 € ($337) per ton at the time of the
capital costs ranging from $0.02 to $1.10 kg− 1 feedstock and higher article) (Voutilainen, 2020). It should be mentioned that sucrose is
production costs that were reported to be between $0.10 to $3.37 kg− 1 cheaper than glucose which is commonly employed in cultivation pro­
sugar (Cheng et al., 2019). Thus, it can be expected that using cesses (see above). The fermentable feedstock was then used for biomass
fermentable sugars derived from lignocellulose will increase the costs of (Candida utilis, Paecilomyces variotii, Fusarium venenatum) and precision
precision and biomass fermentation. However, a recent report that (microbial egg protein by Trichoderma reesei) fermentation. Taking into
focused on lignocellulosic material that is commonly found in the USA account the protein content of the individual sources, the obtained
concluded that the derived fermentable sugar costs range from $320 to minimum protein selling prices (MPSP - i.e., what would be the price to
$550 per metric ton, and if only glucose was considered the associated be at least profitable based on the required costs) was 5160 €/ton
costs were $550 to $880 per metric ton. This was in the same range as ($5615) (Paecilomyces variotii), 6549 €/ton ($7018) (Fusarium ven­
commonly employed DE95 syrup (which is derived from starchy crops) enatum), 7311 €/ton ($7127) (Candida utilis), and 9007 €/ton ($9802)
with $400 per metric ton (GFI, 2023). Based on this, the authors ranked (microbial egg protein) €/ton protein. The authors concluded that “[…]
corn stover, soy straw, and sugarcane bagasse as especially promising comparison between current commercial products and MPSPs impli­
lignocellulosic materials to produce fermentable sugars. cates considerable sales margin even at current technology and strains.”.
To overcome cost constraints several research and development Thus, under the assumptions used, the process was commercially
projects are still being pursued. For example, current efforts include feasible but it excluded certain parameters such as final product
converting at least 90% of glucan to glucose and 90% xylan to xylose at formulation. More studies are needed that confirm the reported results,

Table 2
Selected studies that used food sidestreams to produce microbial biomass or specific compounds.
Sidestream Microorganism Other Media Components Final Product Reference

Yarrowia lipolytica CBS 2070 and

Single cell protein (Peterson et al.,
Cocoa Fatty Acid Distillates Rhodosporidium toruloides ATCC Yeast extract, peptone, tween 80
and oil 2023)
10788
Brewer's spent grains, malt spent
P. ostreatus (Aggelopoulos et al.,
rootlets, cheese whey, molasses, Pleurotus ostreatus –
mycelium + Media 2018)
orange, and potato pulps
Glucose, peptone, meat extract, yeast extract,
Lactobacillus delbrueckii subsp. (Akermann et al.,
Brewers' spent grain liquor Tween 80, K2HPO4, sodium acetate, Lactate
lactis 2020)
MgSO4⋅7H2O, and MnSO4⋅H2O
Chlorella sorokiniana (Pruksasri et al.,
Hydrolyzed rice bran Chlorella sorokiniana Bold's Basal Medium
biomass 2019)
Cheese whey Kluyveromyces sp. Without or with selected supplements Ethanol (Tesfaw, 2023)
(Souza Filho et al.,
Potato protein liquor Rhizopus oryzae Invertase Fungal biomass
2017b)
Fruit pomaces (apple, pomegranate and
Aspartic acid, NH4NO3, KH2PO4, MgSO4⋅H2O (Ahlborn et al.,
aronia), leaf spinach and beet Pleurotus sapidus Mycelium biomass
and 1 mL trace element 2019)
molasses
Leuconostoc mesenteroides MTCC
Sugarcane molasses – Dextransucrase (Sharma et al., 2016)
10508
(Hashempour-
Date Waste Fusarium venenatum Vogel media Mycelium biomass
Baltork et al., 2020)
Beet pulp and potato peel Neurospora crassa not reported Mycelium biomass (Pattillo, 2022)

6
L. Grossmann Biotechnology Advances 73 (2024) 108367

but this is a promising first study showing the potential of using and food production processes as potential feedstocks.
lignocellulosic-derived fermentable sugars for biomass and precision Broadly, the studies that have been published can be categorized
fermentation. based on the microorganism that was used: fungi or lactic acid bacteria.
Besides the potential economic constraints it has to be considered It has to be mentioned that researchers used the sidestreams as feed­
that the released feedstocks consist of different types of sugars, namely stocks for the cultivation but not necessarily to only obtain microbial
C5 and C6 monosaccharides (Farias et al., 2022). This is different biomass or specific compounds. Often researchers also produced a
compared to the glucose-only media that is typically used as a feedstock hybrid product that consists of the microbial biomass and the sidestream
and not every organism can utilize all the different monosaccharides (Raak et al., 2023; Sommer et al., 2023). Fungi have been predominant
that are released from hemicellulose during the hydrolysis step. For candidates for transforming sidestreams because of their high hydrolysis
example, conventional Saccharomyces cerevisiae strains are not able to activity and ability to grow on different substrates. Studies used side­
metabolize C5 sugars such as xylose, and metabolic engineering is streams such as cocoa fatty acid distillates (Peterson et al., 2023),
required to enable the organism to also utilize C5 sugars (Kobayashi brewer's spent grains, malt spent rootlets, cheese whey, molasses, or­
et al., 2018; Lagos et al., 2023). Another constraint is the potential ange, and potato pulps (Aggelopoulos et al., 2018), brewer's spent grain
presence of inhibitors, as discussed before. There is little information liquor (Akermann et al., 2020), rice bran (Pruksasri et al., 2019), cheese
about how the organisms that are commonly used for precision and whey (Risner et al., 2018; Tesfaw, 2023), potato protein liquor (Souza
biomass fermentation grow when certain inhibitors that are formed Filho et al., 2017b), fruit pomaces (apple, pomegranate and aronia), leaf
during the pretreatments are present. This could be a major constraint spinach and beet molasses (Ahlborn et al., 2019), beet pulp and potato
and would also hamper some economic assumptions. peel (Pattillo, 2022), sugarcane molasse (Sharma et al., 2016), and date
It should also be highlighted that lignocellulosic material can be as well as other fruit sidestreams (Hashempour-Baltork et al., 2020;
directly transformed into edible biomass through fungal digestion. Many Kumar et al., 2023; Reihani and Khosravi-Darani, 2018).
commercial operations that produce edible fungi are already using such The cultivation parameters and main results are summarized in
substrates (Ajmera, 2022). Instead of letting fungi produce cellulase and Table 2. A few results are highlighted in the following, but it should be
hemicellulase enzyme mixtures that are then separated and used to kept in mind that each sidestream requires thorough analysis for the
digest lignocellulose, fungi can be directly grown on lignocellulosic availability of fermentable sugars and the selection of suitable micro­
biomass and the evolving mycelium and/or the fruiting bodies can be organisms. Peterson et al. (2023) employed the oleaginous yeasts Yar­
harvested for consumption (Scholtmeijer et al., 2023). Biomass such as rowia lipolytica CBS 2070 and Rhodosporidium toruloides ATCC 10788 on
compost, straw, or sawdust can be fermented by edible fungi through a media that contained cocoa fatty acid distillates (sidestream from
solid state or submerged fermentation. While in principle this is an deodorization or degumming processes). This sidestream mainly con­
established principle, in-depth studies are missing that show the proof of sists of 95% lipids, which makes it a difficult substrate because of the low
concept of this approach especially when compared to the enzymatic water solubility. To increase the solubility, the authors dispersed the
route. distillate in ethanol to increase the mass transfer. This yielded overall
On a final note, emissions of cellulosic sugars have been reported to favorable mass yields of cell dry weights of up to 40.7 g/L for
be between 0.20 and 2.50 kg CO2-eq. per kg sugar, depending on the R. toruloides with volumetric productivities of 3.4 g/L h− 1 when the
source (only forest sources have been considered in this study) and medium contained 20 g/L of distillates. These cultivation conditions
pretreatment (Table 1). This can be higher than first-generation sugars resulted in a microbial biomass composition of 26% lipids and > 20%
(starch or sugar crops derived) with 0.34–1.30 kg CO2-eq. per kg sugar protein for R. toruloides, whereas Y. lipolytica biomass contained 47%
(similar findings have been reported for land use). A recent study found protein with a lipid content of around 20%. Thus, cocoa distillates could
also higher emissions for a yeast-based SCP (for feed) production based be a useful supplement to produce lipid and/or protein-rich biomass
on oat fiber-derived fermentable sugars. Greenhouse gas emissions were with such oleaginous yeast strains.
205–754% higher compared to reported values for yeast protein Another study investigated fruit pomaces (apple, pomegranate, and
concentrate, soy protein concentrate, insect meal, and methanotrophic aronia), leaf spinach, and beet molasses as potential carbon sources. The
bacteria, but required 61% less land compared to soy protein concen­ carbohydrate content was adjusted to 15 g/L and the medium was for­
trate (Kobayashi et al., 2023). However, when first-generation sugars tified with l-aspartic acid, NH4NO3, KH2PO4, MgSO4, and a trace
derived from wheat and corn flour were modeled in the process, the element solution. Seven different basidiomycetes were investigated as
emissions and land use were lower for the media that contained potential consumers of the sidestreams. All of them were able to grow on
fermentable sugars from wet processed oat fibers. Thus, the overall apple pomace but no growth was observed on the other sidestreams.
process was high in emissions independent of the sugars used, which Depending on the fungi, crude protein contents reached up to 25.4% in
reveals the potential for further optimizing fermentation processes. dry matter and the fungal biomass concentration achieved was up to
Nonetheless, careful selection of the lignocellulosic raw material is 15.3 g/L. The reported protein content is lower compared to other
required that yields high sugar contents, requires little land, and has low commercialized species used for mycoprotein production such as Fusa­
emissions (Bello et al., 2021; Kobayashi et al., 2023). rium venenatum, which reaches up to 45% of protein in dry matter
(Finnigan et al., 2019). Moreover, the performance of the cultivation
2.1.2. Food and agricultural sidestreams approach is unclear because only experiments with the sidestreams were
Lignocellulosic materials are a globally abundant feedstock but carried out. Future studies need to evaluate the growth performance of
commonly have to be transformed into fermentable sugars first before the fungi under standardized conditions (e.g. with glucose media)
they can be employed as carbon sources in biomass and precision compared to the sidestreams to fully understand the potential. The
fermentation. An alternative feedstock is sidestreams from agricultural economic viability of such processes often depends on the operational
and food production that already contain available fermentable sugars costs that depend on the price of the feedstock (sidestream) and the
(Table 1). These sidestreams can directly be used as a media ingredient growth performance compared to using a standardized mono/
or can be used as a supplement to lower the required amount of added disaccharide-based feedstock (Makepa et al., 2023).
glucose or other sugars in the formulation. There is a high potential of The so far mentioned studies aimed at generating fungal biomass that
using such sidestreams for generating edible microbial biomass and in- can be transformed into a food ingredient or can directly be consumed as
depth analyses of individual food production processes are required to food. One study used a different approach and used a sidestream to
identify sidestreams that could be used for such purposes (Cardoso Alves produce extracellular dextransucrase. This enzyme catalyzes the for­
et al., 2023). The following paragraphs highlight a few studies that have mation of prebiotic oligosaccharides in the presence of an acceptor by
explored the possibility of using different sidestreams from agricultural transferring the glucosyl moiety of sucrose onto the acceptor to form

7
L. Grossmann Biotechnology Advances 73 (2024) 108367

Fig. 2. Process flow of producing single cell proteins (protein-rich microbial biomass) for food applications using hydrogen gas fermentation from water electrolysis
(left) and acetate fermentation using CO2 electrolysis (right). Adapted from (Hann et al., 2022; Woern and Grossmann, 2023) under CC BY 4.0 Deed and data taken
from (Järviö et al., 2021; Sillman et al., 2019). Icons from [Link].

oligosaccharides (Tingirikari et al., 2017). Sharma et al. (2016) culti­ high operational costs in the mycoprotein scenario. Also, biogas was not
vated Leuconostoc mesenteroides MTCC 10508 on sugarcane molasse, a profitable scenario because of the low solids concentration in the li­
which contains around 50% sugars that are too costly to extract. The quor. However, the authors assumed that the mycoprotein will be sold as
lactic acid bacteria successfully produced the extracellular dex­ fish feed, which has a lower value compared to food ingredients. A
transucrase using molasse as a media ingredient. The crude enzyme product with a higher value may therefore make the approach profit­
extract showed slightly lower sucrose conversion rates compared to a able, but more studies are needed to confirm this.
purified enzyme. The authors hypothesized that the extract may contain A last challenge that has to be mentioned is the potential presence of
some inhibitors that slow down the reaction and further purification inhibitors that may limit the growth of many species. As mentioned
might be needed. above, mainly fungi have been cultivated on media that contained
In conclusion, these studies show that different sidestreams can be sidestreams and surprisingly not all fungi could utilize these media
used to obtain either microbial biomass or specific compounds through (Ahlborn et al., 2019). Compounds that are present in many plants such
microbial production, but especially economic assessments are missing as phenolic acids, flavonoids, stilbenes, and tannins are known to have
at the moment that evaluate the potential of using sidestreams instead of antimicrobial activity and the lignocellulose in many sidestreams is also
refined ingredients. not readily accessible for microorganisms (see section 2.1) (Takó et al.,
2020). Thus, an ideal sidestream that can be used as an energy source for
[Link]. Challenges. When employing sidestreams from food and agri­ biomass and precision fermentation should (i) consist of high amounts of
cultural production, the generated emissions have to be carefully eval­ soluble fermentable sugars, (ii) have consistent availability and
uated. The study of Kobayashi et al. (2023) highlighted the composition, (iii) be low cost, (iv) does not contain any inhibitors or their
environmental burdens of transporting wet sidestreams for further removal should be associated with low costs, and (v) should be easy to
processing. Typically, transporting wet sidestreams over long distances separate from the microbial biomass after completion of the cultivation
(>100 km) emits more emissions than drying. Local transformation of process without leaving adverse residues.
the sidestreams would therefore be beneficial to minimize transport or
drying-related emissions but this highly depends on the availability of 2.2. Gaseous feedstocks for chemoautotrophs
the sidestream, which is another important criterion. The composition
as well as the reliable availability that fits respective production Most microorganisms are cultivated on solid substrates. These solid
schedules has to be ensured. substrates are either solubilized (e.g., glucose solution) or a solid-state
Another factor that has to be considered is the economic viability of fermentation is carried out (e.g., agar). A third approach - which until
using sidestreams. Souza Filho et al. (2017a) compared three different now only received little attention - is the use of gaseous feedstocks to
methods of valorizing potato liquor, which is the liquid sidestream from deliver biochemical energy instead of using solid substrates. These
potato starch processing. The authors evaluated the capital and oper­ alternative feedstocks are reactive gases, such as methane, hydrogen, or
ating costs of transforming the liquor into mycoprotein (airlift biore­ carbon monoxide (Woern and Grossmann, 2023) (Table 1). Especially
actor setup), biogas, or as a concentrated fertilizer. None of the scenarios hydrogen (to cultivate hydrogen-oxidizing bacteria) and methane (to
had a ‘Net present value’ (i.e., were profitable) during a 15-year period. cultivate methanotrophs) have already been successfully implemented
This was related to the high capital costs in the fertilizer scenario and the as feedstocks to produce food and feed ingredients (Cantera et al., 2018;

8
L. Grossmann Biotechnology Advances 73 (2024) 108367

García Martínez et al., 2022; Marcellin et al., 2022). The following Table 3
paragraphs will briefly describe the technology. For a more in-depth Nitrogen and protein-rich agriculture and food processing sidestreams. Adopted
review, the reader is referred to Woern and Grossmann (2023). from (Prandi et al., 2019).
Hydrogen-oxidizing bacteria exploit the ‘Knallgas’ reaction to obtain Sidestream Total nitrogen (g/kg Total proteins (% dm)
biochemical energy for their metabolism (Brigham, 2019). The overall dm)
chemical reaction is: Apple cake 5.48 3.11
Apple pomace 6.94 3.99
6H2 + 2O2 + CO2 →CH2 O + 5H2 O Whole apples 3.82 2.23
Whole pears 3.63 2.09
Based on this equation, 3 types of gases have to be sparged into a
Berry waste 12.13 6.51
bioreactor to grow hydrogen-oxidizing bacteria together with a nitrogen Grape pomace 20.71 11.68
and phosphorous supply: hydrogen, oxygen, and carbon dioxide (Fig. 2). Orange peels 17.06 9.67
The hydrogen-oxidizing bacteria assimilate the supplied H2 and split it Sea buckthorn pulp spent grain 24.25 13.11
by the action of hydrogenases into two electrons and two protons that Sour cabbage 9.85 5.87
Fresh cabbage 11.71 6.52
are then either used in the electron transport chain to regenerate ATP or Sour cucumber 13.74 7.88
directly to reduce NAD+ (Woern and Grossmann, 2023). Thus, the Belgian endive root 12.95 6.86
metabolism of hydrogen-oxidizing bacteria does not require any Belgian endive leaves 29.15 17.35
reduced carbon source such as glucose or other sugars. The main Hop 47.29 24.13
Leek leaves 33.63 19.35
advantage of this is twofold. First, because no agricultural input is
Onion hulls 13.00 7.25
required, this cultivation approach provides a value chain that over­ Onion slices 4.73 2.75
comes the ‘food vs. bioreactor’ challenge, i.e., that food is produced Parsley 29.68 17.03
using agricultural land that is then fed into a bioreactor instead of being Pea hulls 5.18 3.13
directly consumed. Cultivating hydrogen-oxidizing bacteria establishes Potato skins 18.79 10.85
Potato selected 14.91 8.54
an additional value chain that enables more food to be produced without Fresh pumpkin 16.21 9.20
competing for agricultural land (Järviö et al., 2021). Second, low- Sabal 10.97 6.06
emission renewable energy can be used to generate green hydrogen, Sugarbeet flakes 14.73 8.54
which results in overall favorable emissions for the production of Tomato skins 17.26 9.90
1 Topinambur 9.07 4.97
hydrogen-oxidizing bacterial biomass of 1.2 kg CO2eq kg−Product for a
1 Barley rest 16.03 9.29
wind-energy based value chain and 2.2 kg CO2eq kg−Product for a solar- Malted barley germs 51.47 29.11
based value chain (Järviö et al., 2021; Sillman et al., 2019) (Fig. 2). Brewing cake 51.23 30.42
Using hydrogen as a feedstock is, therefore, a promising solution to Olive pomace 12.86 7.67
move towards agricultural-independent biotechnology. Pumpkin kernel cake 101.80 53.04
Rapeseed presscake 43.13 24.40
At present, mainly two protein-rich hydrogen-oxidizing bacterial Sea buckthorn seed 45.37 23.14
species are being investigated as promising candidates for human con­ Hard cheese 67.96 41.84
sumption: Xanthobacter VTT-E-193585 and Cupriavidus necator H16. Soft cheese 64.87 39.77
Both have a high protein content of 72% and 88%, respectively Milk whey 22.41 13.70
Yoghurt 30.49 19.18
(Holmström and Pitkänen, 2021; Modica et al., 2023). The cultivation is
Mushrooms 46.24 26.36
carried out by sparging 70–85% H2, 6–20% O2, and 8–11% CO2 into a Beer yeast 67.56 38.34
bioreactor either at ambient or at elevated pressure (typically below 0.4
MPa) to increase gas solubility (Yu and Munasinghe, 2018). The media is
supplemented with a nitrogen source and other essential nutrients directly be used to fuel a process based on hydrogen-oxidizing bacteria.
required for growth (Woern and Grossmann, 2023). To prevent a The proposed advantage of producing CH4 instead of using H2 gas
possible ignition, the gas mixture supplied contains either oxygen con­ directly is the lower associated storage and transport costs of CH4
centrations that are below the flammability limit (around 5%) or the compared to H2, which could lower the overall costs of the gas supply
cultivation is carried under explosion-proof conditions. The gases are chain (Krol and Lenz, 2020). However, actual studies to prove this claim
supplied continuously throughout the cultivation process and the are missing and will also highly depend on the supply chain of the
biomass is harvested and processed into food ingredients. Safety as­ gaseous feedstock (i.e., local H2 production vs. delocalized H2 produc­
sessments and food approvals for the bacterial biomass have been car­ tion with transport).
ried out or are underway ([Link], 2023; Modica et al., Commercialized products that are based on methanotrophs include
2023). FeedKind®, which consists primarily of the methanotroph Methylococcus
A similar approach is employed for methanotrophic bacteria, but capsulatus (Bath) (FDA, 2021). It is produced in a U-loop bioreactor and
methanotrophs use methane as their sole carbon and energy source: the non-metabolized methane is burned to generate heat for the spray-
drying process to dry the biomass (Cumberlege et al., 2016; Nguyen
CH4 + NADH + H+ + O2 →CH3 OH + NAD+ + H2 O et al., 2023; Olsen et al., 2009). The resulting powder has a protein
This means that no CO2 gas needs to be supplied to the bioreactor, content of about 70%, with 10% being fat, and 1% ash (FeedKind, 2024;
but methane and oxygen (or air) suffice as gas supply. The formed Olsen et al., 2009). It has to be mentioned that also 3 other heterotrophic
methanol is subsequently further oxidized to formaldehyde, which is organisms are present in the biomass. These metabolize the organic
eventually oxidized to CO2 (Woern and Grossmann, 2023). Methano­ acids excreted by Methylococcus capsulatus and other compounds that are
trophs, therefore, release CO2 gas during cultivation, which is in contrast released through cell lysis, as well as reduce the risk of contamination
to hydrogen-oxidizing bacteria. A major challenge in this approach is to with other microorganisms (FDA, 2021). The present organisms are
identify a sustainable methane source because the methane is currently Cupriavidus sp. (6–8% of total cell count), Aneurinibacillus danicus (< 1%
typically supplied by natural gas (Valverde-Pérez et al., 2020). Efforts to of total cell count), and Brevibacillus agri (< 1% of total cell count) (FDA,
produce ‘e-methane’ (also called synthetic methane) are currently un­ 2021). Calysta (Menlo Park, CA, USA) scaled the production of this
derway that rely on similar principles as the Sabatier method (4H2 + ingredient to 20,000 t per annum per facility (Calysta, 2022). Other
CO2 → CH4 + 2H2O) and ultimately require the supply of H2 and CO2 gas companies that use similar technologies include, for example, Unibio
(Tokyo Gas, 2022). This means that the required gases could also (Roskilde, Denmark) (Linder, 2023).

9
L. Grossmann Biotechnology Advances 73 (2024) 108367

2.2.1. Challenges microorganism, but most commonly ammonium salts, nitrates, nitrites,
Both processes - cultivation of hydrogen oxidizing bacteria and and urea are used (Marsh, 1984; Matsudo et al., 2009; Rodrigues et al.,
methanotrophs – rely on the availability of green hydrogen to ensure 2010). As mentioned in the previous sections, all of these compounds are
low emissions. In 2021, only 0.04% of H2 was produced from electricity initially derived from the Haber-Bosch process that uses fossil fuels. In
(IEA, 2022). Thus, to scale this novel cultivation technology, the pro­ this section, possible alternative nitrogen sources will be discussed.
duction of green H2 has to be increased considerably to ensure that
enough H2 gas is available at a competitive price (see also section 3.2.1 3.1. Food and agricultural sidestreams
for a more in-depth discussion on this). Moreover, the cultivation pro­
cess relies predominantly on electricity, and electricity prices will The type of nitrogen fertilizer that can be used to produce foods is
therefore have a major impact on the production costs. The International restricted by regulations and ethical considerations. Certain laws pro­
Renewable Energy Agency (IRENA) outlines key components to achieve hibit the use of specific nitrogen-containing compounds, such as pro­
competitive hydrogen production costs of approximately $1/kg, as per cessed animal protein that was heavily regulated until recently due to
current projections. It's worth noting that hydrogen derived from natural BSE concerns (EC, 2021). Moreover, not every nutrient stream can be
gas currently ranges from $0.98 to $2.93/kg (IRENA, 2020; Schelling, utilized for ethical reasons. Consumers would most likely reject micro­
2023): bial biomass that is cultivated using nitrogen recovered from digestate, a
well-researched source of nitrogen (Beckinghausen et al., 2020). This
• 80% reduction in electrolyzer cost; limits the types of sidestreams that can be utilized to recover nitrogen for
• Electricity cost from 53 to 20 USD/MWh; media formulation mainly to resources from food and agricultural
• Electrolyzer efficiency from 65% to 76% (lower heating value); processes.
• Full load hours from 3200 to 4200 h; Food and agricultural sidestreams can have a considerably high
• Increase the lifetime of electrolyzers from 10 to 20 years; and content of nitrogen and, consequently, protein (Table 1). In an extensive
• Weighted average cost of capital from 10% to 6%. study, Prandi et al. (2019) measured the nitrogen and protein content of
39 different food sidestreams including fruit, vegetable, grain, oilseed,
This suggests that the technology of employing green H2 as a feed­ dairy, and fungal processing byproducts. The total nitrogen varied from
stock for cultivation will become considerably cheaper in the future 3.63 g/kg dm in whole pears to 101.80 g/kg dm in pumpkin kernel cake,
because globally there is a trend towards the production of cheap green which translated to protein contents of 2.09% and 53.04% in dm,
H2, which will also be beneficial for this cultivation technology. More­ respectively (Table 3). In another recent analysis, the authors investi­
over, the costs of producing renewable electricity have dropped gated the sidestream potential from the major crops produced in North
considerably over the last decade and are expected to continue to America (Canada, Mexico, and the United States) (GFI, 2023). Three
decrease. For example, electricity costs produced by photovoltaic different categories were established: (i) protein concentrates as food
dropped by 89% and that from onshore wind by 70% from 2009 to 2019 ingredients, (ii) protein hydrolysates for fermentation or cultivated meat
(Roser, 2023). With the increased deployment of renewable energy media, and (iii) sugars derived from lignocellulosic materials for
around the world, ample green electricity could fuel the production of fermentation media. The authors identified nine sidestreams with high
chemoautotrophic bacteria that grow on H2. Another option is ‘gold’ protein content: barley-based brewers spent grain, canola meal, corn-
hydrogen that is harvested from natural sources. Extensive exploration based distiller's dried grains with solubles, corn gluten meal, rice bran,
efforts are currently underway to understand, quantify, and extract soy meal, tomato pomace, wheat germ, and wheat gluten. The report
hydrogen that is naturally produced within the earth's outer layers incorporated a cost assessment to evaluate the expenses linked to pro­
through serpentinization, radiolysis, or directly from the earth's core cessing the raw materials for the release of amino acids and peptides,
(Hand, 2023; Osselin et al., 2022). These natural H2 sources could which makes them more easily accessible to microorganisms. The inte­
potentially be vast, but only future data will show how much gold H2 is grated ranking was based on volume forecast, unit costs, essential amino
actually present and whether it can be economically harvested. acid content, and environmental impact. Based on this ranking, the most
Another challenge is the lack of information about the use of these promising sidestreams were: soy meal > soy meal protein concentrate >
ingredients in foods and their nutritional properties. As of now, there is corn gluten meal > corn distiller's dried grains with solubles > tomato
no published data on the downstream processing to obtain functional pomace.
protein ingredients using hydrogenotrophic or methanotrophic biomass. Especially soy meal is an attractive source to generate a nitrogen-rich
Similarly, the physicochemical and functional properties of such a feedstock because of its high production volume of >250 million metric
protein ingredient are unknown. More studies are urgently needed that tons in 2022/2023 (Statista, 2023). Coupled with a high protein content
demonstrate the potential benefits of these novel ingredients for food (43.8–49.9% in dm (Banaszkiewicz, 2011)) and a DIAAS of 87, soybean
applications beyond their favorable environmental sustainability. meal is an interesting source to produce nitrogen feedstocks for biomass
A last challenge is that only specialized bacteria can use oxidizable and precision fermentation (Van Den Berg et al., 2022). It has to be
gases as their energy source. Recent research demonstrated the use of mentioned that such protein quality indices have been developed based
electrolyzer technology to obtain acetate from H2O and CO2, which is a on the needs of the human body and might not reflect the needs of the
more versatile media ingredient and was able to be utilized by organisms respective cultivated organism. However, because of the high quality
such as Saccharomyces cerevisiae, Chlamydomonas reinhardtii, pearl oys­ and the low price of soybean meal, it can be argued that soybean meal
ter, enokitake, elm oyster and blue oyster mushroom (Hann et al., 2022) might rather be used directly for human consumption instead of pro­
(Fig. 2, right). Thus, there is a tremendous potential to use electrolyzer cessing it first through a fermentation process. Other feedstocks with
technology to obtain both gaseous and non-gaseous feedstocks for lower potential for direct human consumption such as corn gluten meal,
cellular agriculture. distiller's dried grains with solubles, and tomato pomace (among others)
might be better suited to generate such nitrogen fertilizer for cultivation
3. Sustainable nitrogen sources media. These sidestreams generally have a lower protein quality and are
therefore less suited for direct human consumption. The lower protein
Nitrogen is an integral component of every cultivation media. Mi­ quality could be overcome by blending approaches that can be tailored
croorganisms can utilize an astonishing variety of nitrogen compounds to the needs of specific organisms. For example, distiller's dried grains
ranging from dinitrogen, ammonium, and nitrate to more complex with solubles is relatively low in lysine, glutamine, and tryptophan. To
organic compounds such as urea and amino acids (Magasanik, 1982). compensate these limiting amino acids, it could be blended with canola
The type of nitrogen that can be assimilated depends on the type of meal (relatively rich in lysine) and corn gluten meal (relatively rich in

10
L. Grossmann Biotechnology Advances 73 (2024) 108367

tryptophan) (GFI, 2023). adjusted to the desired value, and the generated hydrolysate is auto­
To generate a suitable nitrogen cultivation fertilizer from the un­ claved before the cultivation process. It has to be noted that each of these
treated sidestream feedstocks, a hydrolysis step is typically employed to enzyme preparations has different optimum reaction temperatures and
release amino acids and peptides. These protein fractions are then pH values. Thus, the raw material has to be adjusted to the respective
metabolized by the microorganism. Three main approaches have been optimum conditions of the enzyme.
employed to hydrolyze protein-rich raw materials to release amino acids The third approach utilizes the hydrolyzing activity of fungi to break
and peptides to transform sidestreams into nitrogen feedstocks for down different sidestreams and plant materials. Fungi are generally
cultivation: acid, enzyme, and fungal-assisted enzyme hydrolysis. First, known to employ extracellular digestion to release small molecular
acid hydrolysis employs inorganic acids such as oxalic acid, sulfuric weight compounds that can be taken up by their cells using osmotrophy
acid, and hydrochloric acid (1–6 M depending on acid, temperature, and (Jones et al., 2011). This type of foraging can be exploited to cultivate
time) to chemically hydrolyze the peptide bonds and release amino acids fungi on raw materials that are digested by the fungi through extracel­
(Dai et al., 2019; Zhang et al., 2022). Samples are heated to elevated lular enzymes. For example, Aspergillus awamori and Aspergillus oryzae
temperatures, sometimes above 100 ◦ C (Brock et al., 2019; Krull et al., have been used to hydrolyze mixed food waste, and the total nitrogen
2020). After the hydrolysis, a liquid-solid separation is typically carried released after 24 h submerged cultivation was 280 mgFree Amino Nitrogen
out (for example by simple centrifugation) and the supernatant is L− 1 when both fungi were used in a co-culture. Only slightly higher
collected, neutralized, and sterilized before the incorporation into the values (291 mgFree Amino Nitrogen L− 1) were obtained when commercial
cultivation process (Pleissner and Venus, 2014). Moreover, the nitrogen enzymes were used instead (Sun et al., 2014). In a similar study, bakery
concentration can be tailored to the needs of the microorganism by waste and food waste were hydrolyzed with the same fungi (Lau et al.,
measuring the total nitrogen content of the resulting hydrolysate using 2014). The process consists of initially generating an inoculum culture
standardized methods such as Dumas or Kjeldahl (McClements and by solid-state fermentation. This inoculum is used to initiate the hy­
Grossmann, 2022). A disadvantage of this method is the use of highly drolysis step in a submerged cultivation approach. After the fermenta­
concentrated acids, the need for neutralization, and the high salt content tion process, a liquid/solid-separation is carried out to remove the fungi
in the final supernatant. For these reasons, other authors also explored from the solubilized compounds, which are then used as fermentation
the use of enzymes for the production of hydrolysates. substrates for the target organism (Sun et al., 2014).
Proteases have been used to hydrolyze a variety of different proteins Acid and enzymatic hydrolysis have been successfully employed to
both from animal and plant origin (Tavano et al., 2018). To hydrolyze transform a variety of different plant raw materials into nitrogen-rich
sidestreams for the production of a nitrogen fertilizer, different and fertilizers including rapeseed meal (Wongsirichot et al., 2022), dis­
mostly commercial enzyme preparations have been used (Brock et al., tillers' dried grains with solubles (Krull et al., 2020), corn gluten (Brock
2019; Krull et al., 2020; Zhang et al., 2022). Protease preparations such et al., 2019), sunflower meal (Brock et al., 2019), rape cake (Brock et al.,
as Alcalase® (Novozymes, Copenhagen, Denmark), Fermgen™ 2019), wheat gluten (Brock et al., 2019), cottonseed meal (Zhang et al.,
(DuPont™ Genencor, Rochester, NY, USA), Flavourzyme® (Novozymes, 2022), food waste (Lau et al., 2014; Sun et al., 2014), bread waste
Copenhagen, Denmark), Neutrase® (Novozymes, Copenhagen, (Leung et al., 2012), spent yeast (Ryu et al., 2013), and bakery leftovers
Denmark), Protamex™ (Novozymes, Copenhagen, Denmark), and Pro­ (Pleissner et al., 2014). Most of these mentioned studies aimed at using
tex™ 6 L (DuPont™ Genencor, Rochester, NY, USA) have been tested for fermentation to produce specific compounds such as succinic acid, lactic
their hydrolyzation capabilities of agricultural and food sidestreams acid, and polyhydroxybutyrate. There have been limited studies on
(Brock et al., 2019; Krull et al., 2020). These enzyme preparations are utilizing such sidestreams for biomass production, such as fungi
typically endopeptidases (Alcalase, Fermgen, Neutrase, Protamex, Pro­ biomass, or employing precision fermentation to generate specific types
tex) or mixtures of endo- and exopeptidases (Flavourzyme). of proteins, like β-lactoglobulin. Thus, it is difficult to evaluate the ef­
To carry out the hydrolysis, the plant raw materials are mixed with ficiency of using nitrogen that comes from sidestreams and not from
the enzymes and hydrolyzed for 24–48 h. After the hydrolysis, the pH is purified synthetic nitrogen compounds in these cultivation processes.

Fig. 3. Overview of basic electrolyzer working principles and green ammonia production process based on hydrogen produced from renewable energy (e.g., solar).
Data taken from (Mayer et al., 2023; Sapountzi et al., 2017; Smith et al., 2020). Icons from [Link]. Abbreviations: TRL = technology readiness level, alkaline
water electrolysis (AWE), polymer electrolyte membrane water electrolysis (PEM WE), and solid oxide water electrolysis (SOE). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

11
L. Grossmann Biotechnology Advances 73 (2024) 108367

However, the current body of literature shows that the conditions of the therefore expensive to collect. Strategies should therefore be developed
hydrolysis have a considerable impact on the production rate. Brock that incorporate more centralized sidestreams that do not require the
et al. (2019) demonstrated that when different food and agricultural pick up of small quantities at many different locations. Third, it has to be
sidestreams were only ‘mildly’ hydrolyzed (pH 1, 121 ◦ C for 20 min) the acknowledged that one advantage of ammonia produced from the
lactic acid concentrations and productivities were considerably lower Haber-Bosch process is its purity. Agriculture and food sidestreams may
compared to the use of yeast extract as a reference. However, when the contain compounds that affect the growth of the microorganism or have
hydrolysis was carried out using concentrated 3 M sulfuric acid at 110 ◦ C to be removed from the final product or biomass after the cultivation
for 24 h (which released the highest concentration of free amino acids), process. Thus, sidestreams need to be standardized to ensure optimum
the lactic acid production rate was almost similar among the different growth rates and yields. Lastly, producing food ingredients on side­
feedstocks tested (rapeseed meal, distillers' dried grains with solubles, streams that are considered “waste” by the general public may result in
sunflower meal, rape cake, wheat gluten, and corn gluten) and the low consumer acceptance. Consumer acceptance tests have to be carried
reference at standardized nitrogen concentrations. In addition, the au­ out before the cultivation on such sidestreams is carried out and prod­
thors showed that considerably lower amounts of amino acids were ucts are released to the market.
released when enzymatic hydrolysis was employed. A maximum of 2.9%
of free amino acids was achieved when rapeseed meal was hydrolyzed
3.2. Electrochemical ammonia production
with Flavourzyme or Alcalase compared to 27% when chemical hy­
drolysis was carried out (Brock et al., 2019). Enzymes are typically also
Around 48% of the crops consumed globally are cultivated using
more sensitive to changes in the raw material and the effectiveness
synthetic fertilizers (Editorial, 2023). The main process to produce
might vary depending on the raw material used, which lowers the
synthetic fertilizers is the Haber-Bosch process. The process is energy-
flexibility of the hydrolysis process. This shows that chemical hydrolysis
intensive, and it is estimated that it uses 1–2% of total global energy,
can be advantageous but that the degree of free amino acids needs to be
3–5% of the globally produced natural gas and is, therefore, also
carefully monitored and adjusted according to the respective cultivation
responsible for 1–3% of the total CO2 emissions (Kyriakou et al., 2020;
process. However, as mentioned before, these results might not be
Soloveichik, 2019; Song et al., 2023). When fertilizers are used in
directly transferable to other microorganisms and may also depend on
cultivation processes they are inherently more efficient compared to
the target compound to be produced (i.e., biomass or specific proteins).
open field applications because of the closed system, which ensures
targeted application and prevents runoff. In fact, the majority of the
3.1.1. Challenges
emissions created by fertilizers occur after they have been applied to
Production of nitrogen-rich cultivation fertilizers from food side­
cropland due to leaching and decomposition into N2O (Gao and Cabrera
streams has still to be implemented at larger scales. One inherent chal­
Serrenho, 2023). In a bioproduction process, the nitrogen fertilizer is
lenge of utilizing sidestreams is the variability in the composition of the
directly applied to the bioreactors and no runoff occurs, especially if the
sidestreams and the logistic challenges of collecting consistent amounts
media is depleted in nitrogen at the harvesting stage. Nonetheless, the
that suffice the production needs. First, depending on the sidestream –
Haber-Bosch process is an energy-intensive process and relies on fossil
especially food waste – the composition and therefore also the nitrogen
fuels. In an effort to decarbonize the food production chain, several
content can vary. For example, nitrogen in food waste digestate was
technologies have been developed to lower the emissions of the Haber-
reported to range from 1.1 kg/t to 9.6 kg/t (Manu et al., 2021), which
Bosch process.
reveals the inherent variability of food waste as a nitrogen source.
Natural gas (mainly CH4) is used in the Haber-Bosch process as a
However, this might be mitigated if access to specific food sidestreams
hydrogen donor to provide three hydrogen atoms to one nitrogen atom
exists, such as rapeseed meal from a rapeseed oil manufacturing plant.
to form NH3. During this process, the leftover carbon is released as CO2
The availability of such centralized and consistent sidestreams should be
from the process, which emits 1.6 kg CO2/kg NH3 (Capdevila-Cortada,
investigated more in the future as possible feedstocks. Second, side­
2019; Osorio-Tejada et al., 2022). A straightforward approach to lower
streams often occur in small quantities at distant locations and are
the carbon emissions from ammonia production is to use another H2

Fig. 4. Simplified working principles of examples of direct electrochemical and bioelectrocatalytic ammonia synthesis. Adopted from (Hickey et al., 2018; Hu et al.,
2023; Jiao and Xu, 2019) under CC BY-NC 3.0 Deed.

12
L. Grossmann Biotechnology Advances 73 (2024) 108367

source in the process. Hydrogen can be classified as grey (from fossil needed to electrolyze water at 25 ◦ C, whereas only 0.95 V are needed
fuels), blue (from fossil fuels with CO2 capture), green (from electrolysis at 900 ◦ C (Deka et al., 2019)) and is, therefore, promising for large-
using renewable electricity sources), and gold (from natural sources) scale H2 production. The SOE is composed of two porous electrodes
(Stalker et al., 2022). It has been shown that green ammonia (i.e., and a dense ceramic electrolyte that is able to withstand O2− ion
ammonia produced from solar-powered H2 production) exhibits drasti­ migration at elevated temperatures of 600 ◦ C to 850 ◦ C (Hauch et al.,
cally lower CO2 emissions in a cradle-to-gate life cycle assessment: 0.5 2020). Water (steam) is split electrochemically into H2 and oxide
kg CO2/kg NH3 for green ammonia compared to 5 kg CO2/kg NH3 (grey ions (O2− ) at the cathode. After diffusing through the electrolyte to
ammonia), and 1.2 kg CO2/kg NH3 (blue ammonia) (Mayer et al., 2023) the anode, the ions are oxidized to O2 and are released (Wolf et al.,
(Fig. 3). Thus, especially green and gold hydrogen are promising alter­ 2023). This type of working principle is also known as high-
natives to lower the CO2 emissions of the process, but they are not temperature steam electrolysis (He et al., 2023).
without challenges. Of the two, not much data is available yet for gold
hydrogen as the exploitation and evaluation is still work in progress. For The overall aim is to integrate green hydrogen generation into the
this reason, the following discussion will focus on green hydrogen. NH3 production process. The process design includes 4 main parts
Green ammonia is derived from green hydrogen (Chehade and (Smith et al., 2020) (Fig. 3): (i) hydrogen is produced from water using
Dincer, 2021). At present, hydrogen is produced by steam methane renewable energy, (ii) N2 is purified from air by pressure swing
reforming, but novel methods to produce ammonia from sustainable adsorption, cryogenic distillation, or membrane separation technolo­
hydrogen include direct electrochemical ammonia synthesis, water gies, depending on scale and purity needed, (iii) both gases are fed into
electrolyzers, bioelectrocatalytic nitrogen fixation, electromagnetic the Haber–Bosch reactor where hydrogen and nitrogen form ammonia at
ammonia synthesis, and photo-electrochemical ammonia synthesis 15–25 MPa and 400–450 ◦ C by employing an iron-based catalyst, and
(Chehade and Dincer, 2021). Of these, especially water electrolyzers, (iv) ammonia is removed in a condenser. This is one of the most prom­
direct electrochemical ammonia synthesis, and bioelectrocatalytic ni­ ising solutions to produce sustainable nitrogen fertilizer in the future.
trogen fixation are being discussed as the most promising solutions Two other solutions that are less mature are discussed in the next
(MacFarlane et al., 2020; Wang et al., 2023). section.
Electrolyzers. Three main types of electrolyzers have been devel­ Direct electrochemical ammonia synthesis. In this approach, the
oped to generate H2 from water that are generally believed to be suited biological nitrogen fixation (see chapter 3.3) is ‘mimicked’ through an
for green ammonia production (Lee et al., 2022b). These electrolyzers electrochemical process that would in principle allow for air and water
require theoretically 0.16 m3 of water per hour and MW of capacity to be used to directly produce ammonia at ambient pressure and tem­
(even though typically double the amount is needed) (Bollmann et al., perature (Lan et al., 2013). The reasons why dinitrogen is so inert are
2023). They are classified based on their operating conditions and numerous and include the high energy gap that exists between the
charge carrier type (OH− , H+, O2− ): alkaline water electrolysis (AWE), highest occupied molecular orbitals and lowest unoccupied molecular
polymer electrolyte membrane water electrolysis (PEM WE), and solid orbitals, the zero net dipole moment, weak polarizability, high ioniza­
oxide water electrolysis (SOE) (Fig. 3): tion energy, low proton affinity, and high bond-breaking energy (Hu
et al., 2023). To overcome this, two techniques have been investigated.
• AWE-type hydrogen generators are one of the most mature electro­ First, the nitrogen can directly be reduced by an electrocatalyst that
lyzer technologies. In this setup, water is split into O2 and H2 at the allows for a direct reaction of the electrons and protons with the N2
anode and cathode, respectively, following this reaction (Keçebaş molecule to yield ammonia (Fig. 4, left). Alternatively, a redox mediator
et al., 2019): - such as Li+ − facilitates ammonia production by forming Li3N as an
Cathode: 4 H2O + 4e− → 2 H2 + 4 OH− intermediate on a copper substrate, which is followed by a number of
Anode: 4 OH− → O2 + 2 H2O + 4e− reactions to form ammonia and the regeneration of the catalyst (Mac­
Total: 2 H2O → O2 + 2 H2 Farlane et al., 2020). Several novel catalysts are currently investigated
The reaction shows that hydrogen forms at the cathode and oxygen such as carbon-based fiber, porous carbon, graphene, nonmetallic
at the anode. The name of this electrolyzer stems from the alkaline element-doped carbon, and graphdiyne (Hu et al., 2023). These catalysts
electrolyte solution in which the electrodes are submerged. Usually, are used in an electrochemical cell, such as the membrane electrode
the solution is composed of 25%–30% potassium hydroxide and so­ assembly (among others that are described in (Hu et al., 2023)). This cell
dium hydroxide as well as sodium chloride as a catalyst (Keçebaş consists of catalyst-coated cathode and anode electrodes and is divided
et al., 2019). The ions present in the alkaline water support the by either a proton exchange membrane or an anion exchange mem­
conduction of electricity to accelerate the electrolysis process. AWEs brane. Throughout the electrolysis process, N2 and H2O are introduced
have the highest technology readiness level and a low capital into the cathode and anode compartments, respectively, to facilitate
expenditure because of the rather cheap catalysts employed (Lee nitrogen reduction and water oxidation, which follows the following
et al., 2022b). reaction under acidic conditions (Hu et al., 2023):
• PEM electrolyzers are the second most common type of electrolyzer Anode: 3H2O → 6H+ + 1.5O2 + 6e−
with a slightly lower technology readiness level than AWEs (Lee Cathode: N2 + 6e− + 6H+ → 2NH3
et al., 2022b). This technology utilizes a solid polymer electrolyte, Bioelectrocatalytic nitrogen fixation. In the second technique, bio­
which allows for a compact design and flexible operation under electrocatalytic nitrogen fixation is carried out by the isolated catalytic
intermittent renewable electricity supply (Lee et al., 2022b). This subunit of the nitrogenase enzyme. The enzymatic nitrogen fixation
hydrogen generator operates by splitting water at the anode into O2, usually requires 16 mol of ATP per mole of N2 (Duval et al., 2013),
protons (H+), and electrons (e− ). The protons subsequently diffuse which is a major drawback when developing such biomimetic processes.
through the proton-conducting membrane to the cathode side of the However, scientists have developed an ATP-independent electro­
electrolyzer. The electrons on the contrary move from the anode enzymatic reduction of N2 by attaching the nitrogenase to an electrode
through the external power circuit. This ultimately drives the reac­ surface. The main hurdle in this approach is the polypeptide surface that
tion and hydrogen is released at the cathode side (Shiva Kumar and has insulating properties and prevents the reaction between the redox-
Himabindu, 2019). active cofactor and the electrode surface (Hickey et al., 2018). The
• SOEs have a technology readiness level of about 5 and are not yet enzyme has therefore to be located very close to the electrode surface
commercially available (Lee et al., 2022b). They are operating on the (14 Ȧ), which can be achieved by, for example, immobilizing the protein
reverse principle of solid oxide fuel cells (Grossmann et al., 2022). on the electrode surface by using cross-linked hydrogels or docking
This electrolyzer technology has a high system efficiency (1.23 V is

13
L. Grossmann Biotechnology Advances 73 (2024) 108367

motifs (Chen et al., 2020). One breakthrough was reported by Hickey which will ultimately determine the costs of operating the electrolyzers.
et al. (2018), who used pyrene to attach a hydrogel onto a carbon However, natural gas prices are also subject to change, either due to
electrode that stabilizes and entraps the recombinantly produced unexpected market fluctuations or because of carbon taxes that have
nitrogenase (Fig. 4, right). The enzyme was encapsulated in a hydrogel been introduced around the world. In fact, as of April 2023, there have
consisting of linear polyethylenimine, pyrene, and ethyleneglycol been 73 carbon taxes or emission trading systems implemented globally
diglycidyl ether, which was coated on a Toray electrode. This approach (World Bank, 2023). As an example, taking the CO2 tax in Sweden from
was able to resolve the ATP bottleneck by delivering the electrons 2021 - which was $137.24 per ton CO2 - increases the price per ton of
directly to the nitrogenase enzyme and enabling electroenzymatic N2 NH3 by $298 when natural gas is the H2 feedstock (Lee et al., 2022b).
reduction to NH3 without the presence of ATP. Thus, this approach Nonetheless, the current cost of producing green ammonia remains
enabled ammonia production using nitrogenase as a catalyst by targeted significantly higher than the baseline scenario using natural gas, even
immobilization and electricity. when factoring in existing CO2 tax pricing schemes. Depending on how
As a final remark, it has to be acknowledged that both of these the costs will develop, current economic assessments indicate that in a
processes are at present at a low technological readiness level of around best-case scenario at high and medium efficiency of the green ammonia
1–3, whereas carrying out the Haber-Bosch process using green production process, the costs will match (high efficiency) or closely
hydrogen from alkaline electrolysis is considered to be almost a mature match (medium efficiency) that of conventional ammonia by 2050. This
technology with a level of 8–9 (Smith et al., 2020). Nonetheless, even will only be successful, if electrolyzers become more efficient, water
with green hydrogen the conventional Haber-Bosch process requires prices decrease (around 21 billion m3 of freshwater will be used for
high temperatures and pressures. Techniques that would allow the optimistic hydrogen economy scenarios, which is <10-times the amount
process to be carried out at lower temperatures and pressures would agriculture is using at present (Beswick et al., 2021; Woods et al.,
therefore be beneficial. 2022)), and levelized costs of renewable electricity continue to decrease
(Lee et al., 2022b). In this assessment, the AWEs and the SOEs exhibited
3.2.1. Challenges the lowest cost burden for green ammonia production. The timeline to
The cost of ammonia is typically around $300–500/t (global value of achieve such a price parity with the conventional Haber-Bosch process
about $250 billion), but traded at a much higher price in 2022 of around highly depends on the CO2 tax and assumes a constant tax until 2050.
$1600 because of the increased natural gas prices (DoE, 2022; Wang Thus, price parity might be reached earlier, if CO2 taxes continue to
et al., 2018). Natural gas typically trades in the USA for industrial ap­ increase over the next decades.
plications between $0.14–0.28 per m3 (eia, 2023). This means that the Other challenges are related to the availability of renewable elec­
price for green H2 has a decisive influence on the success of the green tricity, the design of the electrolyzers, and the materials that are being
ammonia technology because it has to compete with the natural gas used to manufacture them. First, some of the renewable electricity
price. At present, green H2 can be produced for $2.5/kg in the Middle- generation methods – wind and solar – are intermittent by nature.
East by using alkaline electrolyzer technology (Mayer et al., 2023). It Depending on the hydrogen production site, it will be essential to
is believed that other regions, such as the European Union, will require evaluate the available electricity sources, potential electricity storage
at least several more decades to reach this price. Nonetheless, under this technologies, the type of electrolyzer, and whether the electrolyzer is
optimistic scenario (assuming 2.5$/kg), an ammonia price of around installed on- or off-grid (Kojima et al., 2023). It is not well understood
500$/ton can be achieved. how the intermittent nature of many renewable electricity technologies
The study of Mayer et al. (2023) concluded that to break even and be affects the long-term stability of the catalysts and electrodes in typical
competitive, the green hydrogen price has to come down to $1.9/kg to electrolyzer designs. Nevertheless, both, AWEs and PEM electrolyzers
reach $385.8 per ton price of ammonia (Table 1). The authors also noted have been proven to be able to operate under fluctuating electricity
that the hydrogen price will highly depend on the electricity price, conditions but the long-term stability is still somewhat unclear (Ehlers

Fig. 5. Biological nitrogen fixation strategies for green biofertilizer production that can potentially be utilized in cultivation processes. Intracellular route utilizes
diazotrophic bacteria (e.g., cyanobacteria) whereas extracellular route employs metabolic engineering to maximize excreted ammonium (target pathways high­
lighted in red). Adapted from (Ambrosio et al., 2017; Ortiz-Marquez et al., 2014; Sadvakasova et al., 2022). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

14
L. Grossmann Biotechnology Advances 73 (2024) 108367

et al., 2023; Lee et al., 2022b; Voronova et al., 2023). Moreover, it is section will briefly elaborate on how this process could potentially be
expected that the cold startup times especially for AWEs will consider­ exploited to obtain reactive nitrogen compounds for cultivation
ably decrease in the future, which will facilitate its application under processes.
intermittent electricity conditions (Smolinka, 2018). Second, especially The plant-rhizobia symbiosis is a well-designed system in which the
PEM electrolyzer electrodes depend on rare elements such as iridium, plant provides the sugar molecules for the energy-intensive process and
which is the rarest metal in the earth's crust (0.000003 ppm in the earth's the rhizobia delivers the ammonia in return. In a non-symbiotic envi­
crust (Minke et al., 2021)). At present, iridium is used at a density of 2 ronment, the produced ammonia remains within the cell for intracel­
mg cm− 2 in the membrane and projections are that the applied amounts lular processes, which yield amino acids for the bacteria (Wang et al.,
will be reduced to 0.4 mg cm− 2 by 2035 (Minke et al., 2021). When 2023). Two main approaches are utilized to use diazotrophic bacteria as
coupled with heightened recycling efforts, it has been determined that a feedstock for nitrogen fertilizer production (Fig. 5). First, the bacteria
the projected demand for iridium, amounting to 13 metric tons over the are cultivated in a bioreactor setup and the biomass can be applied
next 24 years, can be met, considering the installation of 61 gigawatts of directly as a biofertilizer or the nitrogen/amino acids are released
PME electrolyzer capacity by 2045 (Minke et al., 2021). Lastly, imple­ through a downstream process (Pajčin et al., 2021). This will be referred
menting electrolyzer technology and incorporating it into the Haber- to as ‘intracellular route’. Second, ammonia assimilation in the cell can
Bosch process still needs to be developed on a larger scale, which will be prevented through metabolic engineering and inhibitors to prevent
be challenging, especially for less-developed electrolyzer technologies the cell from producing amino acids from the ammonia. This approach
such as SOE (Bollmann et al., 2023). will therefore be referred to as the ‘extracellular route’.
The first approach - the intracellular route - is only useful if either
3.3. Biological nitrogen fixation high C/low N sidestreams are used that provide the required energy for
the growth of the diazotrophic microorganism or if autotrophic organ­
Many microorganisms rely on reduced nitrogen sources, such as isms are cultivated. If sugars are used for the cultivation process that are
ammonia or urea for their metabolism. However, certain bacteria and obtained from agricultural production that requires fertilizing, the
Archaea are able to fix dinitrogen from the air and transform it into process would be redundant in itself. For this reason, authors investi­
ammonia. These microorganisms have utilized this mechanism to gated using autotrophic organisms such as cyanobacteria and C-rich
recover the inert nitrogen from the air and produce more reactive ni­ sidestreams as energy suppliers (Massey and Davis, 2023; Ospina-
trogen species for hundreds of millions of years (Pankievicz et al., 2019). Betancourth et al., 2020). Cyanobacteria are photosynthetic bacteria
Species from several genera possess the ability to fix nitrogen (known as and nitrogen fixation genes have been identified in a number of species
diazotrophs), such as Azospirillum, Rhizobium, Azotobacter, Klebsiella, (Herrero et al., 2001). The nitrogenase enzyme is very sensitive to ox­
and others (Raffi and Charyulu, 2021) (Table 1). The mechanism of ygen, as described above. The bacteria, therefore, separates the oxygenic
nitrogen fixation in these bacteria is a highly energy-intensive process photosynthesis process from the nitrogen fixation pathway by either
and requires micro-aerobic conditions (Mahmud et al., 2020): carrying out both processes in separate compartments or by utilizing
dark-light cycles to separate both processes using the circadian clock
N2 + 8H+ + 8e− + 16 Mg − ATP→2NH3 + H2 + 16 Mg − ADP + 16 P (Herrero et al., 2001). For example, multicellular Anabaena and Nostoc
are known to possess heterocysts which are specialized cell types to
This reaction is catalyzed by the oxygen-sensitive enzyme nitroge­
carry out nitrogen fixation (Kumar et al., 2010) (Fig. 5, left). On the
nase (which is a two-component enzymatic assembly consisting of the
other hand, the single-cell strain Cyanothece sp. ATCC 51142 produces
heterotetrameric MoFe protein (nitrogenase) and the homodimeric Fe-
glycogen during the presence of light and fixes nitrogen when no
only protein (reductase)). Different organisms typically form a delicate
photosynthesis is carried out (Kumar et al., 2010).
symbiosis with plant roots in which the plant provides protection, low
Cyanobacteria can be cultivated by different cultivation techniques
oxygen concentrations, and energy in exchange for the ammonia pro­
(Grossmann et al., 2019). The most straightforward technique is the
duced by the nitrogen-fixing bacteria in the soil (Mahmud et al., 2020).
raceway pond, which consists of an oval pond with a paddlewheel for
This biological nitrogen fixation is present all over the world and it is
consistent flow and gas mixing. These systems are often open, but can
estimated that through this reaction, 200 million tons of reactive ni­
also be built with roof protection to minimize contamination (Paul et al.,
trogen are produced annually (Mahmud et al., 2020). The following
2021). Such systems have already been successfully applied to obtain a
nitrogen-rich biofertilizer especially in developing countries but also in
other regions of the world. For example, one study investigated using the
Anabaena spp. cyanobacteria grown in raceway ponds with a nitrogen
content of 23.3 mg N/kg (wet basis) as a biofertilizer in carrot produc­
tion (Wickham and Davis, 2023). The cyano-fertilizer consistently ach­
ieved similar yields to a fish-hydrolysate-based fertilizer, which shows
the principal usability of the fertilizer. However, it has to be acknowl­
edged that carrots do not require high amounts of nitrogen fertilizer
because of their low protein content (28.3–68.4 kg N per ha was applied
during the season). In another study, kale, pepper, and maize were
grown with either biofertilizer based on Anabaena sp. or regular urea
fertilizer treatment (Asmamaw et al., 2019). The total nitrogen appli­
cation amounts were 64 (maize) and 100 (kale, pepper) kg N per ha. The
application of the cyano-bacteria fertilizer resulted in similar or higher
plant heights, leaf numbers, and leaf areas. The authors therefore
concluded that especially the dried cyanobacteria (i.e., powder applied
as fertilizer which may enhance N mineralization and thus more effi­
cient fertilization) led to an increase in growth and yield for the inves­
tigated crops even in different soil types. Nevertheless, all these studies
Fig. 6. Potential process flow to purify spent media. Re-using culture media has rely on conventional agricultural applications, and currently, there is a
the potential to minimize lost nutrients. Filtration and purification techniques lack of research on the utilization of a cyanobacteria-based fertilizer in
can be employed to recover nutrients. cultivation processes. Moreover, in these studies, the cyanobacteria

15
L. Grossmann Biotechnology Advances 73 (2024) 108367

have been applied dried or in a liquid formulation directly to the field. 1984), and a commercial production volume of 200,000 L is often used
For a bioreactor application, the ammonia or amino acids have to be (Whittaker et al., 2020). This would require a cyanobacteria cultivation
released before the application to ensure proper N-uptake (Massey and volume of 2,800,000 L (assuming an average N concentration obtained
Davis, 2023). This mineralization process may require a considerable by 30 mg L− 1 for the cyanobacteria cultivation). This means that around
amount of time, which has to be factored in when designing a fertilizer 14 times more cultivation volume is required for the fertilizer produc­
production process based on cyanobacteria. In one study, for example, tion compared to the actual microbial biomass production volume. This
NH+ 4 release peaked after 56 days of mineralization in a fine-loamy, may be a constraint, but it has to be acknowledged that the bioreactor
mixed, mesic Aridic Haplustalf soil, whereas NO−3 release was still in setup for cyanobacteria production is less sophisticated and cheaper to
progress after 140 days (Jama et al., 2023). However, acid or enzymatic build, especially raceway ponds (see above) (Grossmann et al., 2019).
treatments that have been described in section 3.1 may also be used to The same is true for genetically modified diazotrophic bacteria that
release the nitrogen from diazotrophs to obtain fertilizer for cultivation have been bioengineered to release ammonium in the extracellular
processes. approach. As described above, literature data indicates that ammonium
The second approach - the extracellular route - is to promote the concentrations of around 20 mM are feasible using this technique. Using
ammonium release from the cell by either introducing a glutamine similar assumptions (420 mgN L− 1 required in media, 200,000 L pro­
synthetase inhibitor such as methionine sulfoximine or genetically duction volume), this would require a cultivation capacity of around
modifying the cell to excrete the produced ammonium (Fig. 5, right). 300,000 L for the fertilizer production using bioengineered diazotrophs
One method is to induce a point mutation in the gene that expresses the (assuming no concentration step). While this is much less compared to
glutamine synthetase or induce a mutation in the antiactivator NifL (i.e., the production using the intracellular production route, it still requires
prevents nif gene expression and therefore nitrogenase production) that 1.5 times more fertilizer production volumes than actual biomass pro­
regulates the nitrogen fixation depending on the available ammonium duction volumes. This may be a challenge, especially if costly bio­
levels by downregulating the expression of the nitrogenase enzyme reactors are needed for the production.
when enough ammonium is present (Ortiz-Marquez et al., 2014). It was A second challenge is related to the purity of the biofertilizer.
shown that when the NifL antiactivator gene was mutated in Azotobacter Ammonia from the Haber-Bosch process is almost pure ammonia. If the
vinelandii (diazotrophic growth), considerably higher ammonium con­ whole biofertilizer is added as a nitrogen source without extracting the
centrations were released into the surrounding media compared to the nitrogen first, potential inhibition of the production strain and food
wildtype when in addition a glutamine synthetase inhibitor was present safety concerns may arise. The current knowledge on utilizing bio­
(Ortiz-Marquez et al., 2014). By hindering the cell's ability to incorpo­ fertilizer as a nitrogen feedstock in biomass and precision fermentation
rate ammonium into amino acids and disrupting ammonium sensing, approaches is limited and therefore it is unknown how the addition of
this leads to an elevation in the release of ammonium by the cell. unprocessed diazotrophic bacteria to the production strain may impact
The authors subsequently metabolically engineered another step by its growth behavior. Moreover, the inclusion of additional strains
inducing a mutation in the glutamine synthetase gene. This directly beyond the production strain may cause food safety concerns. It has to
stops the ammonium incorporation into amino acids without adding an be ensured that no toxins or allergy risk is introduced, which may
additional inhibitor. And indeed, the mutation resulted in ammonium jeopardize the legal status of the final product. This may be circum­
excretion into the media even without the presence of methionine sul­ vented by utilizing only the ammonia after a downstream process
foximine (Ortiz-Marquez et al., 2014). In the different diazotrophic (intracellular route) or separating the ammonium from the diazotrophic
growth experiments, the ammonium concentration reached >1.5 mM in organism (extracellular route) to obtain a purified nitrogen source that is
the glutamine synthetase mutated strain, and up to 7 mM was achieved standardized to a specific ammonium concentration with standardized
when the NifL antiactivator gene was mutated (and a glutamine syn­ and characterized impurities (if any).
thetase inhibitor was present). It should be noted that the numbers are
not directly comparable because of the different cultivation conditions 4. Sustainable phosphate sources
used. However, under carbon and nitrogen limiting conditions the
strains bearing the glutamine synthetase were more efficient ammonium Phosphorus plays a key role in every organism because it is an in­
producers and were able to sustain the growth of the microalga Chlorella tegral part of every organism's nucleotides (i.e., DNA and RNA), phos­
sorokiniana more efficiently in a co-culturing experiment (i.e., the pholipids (i.e., cell membranes), and energy generation through the
Azotobacter strain delivered the nitrogen for the Chlorella growth) (Ortiz- phosphorylation of ADP to form ATP. Moreover, in some proteins,
Marquez et al., 2014). In a subsequent study, the authors even produced phosphates stabilize the quaternary protein structure such as in the
a double-mutant strain that contained an exogenously inducible pro­ casein micelles (Gonzalez-Jordan et al., 2015). Phosphorous – in the
moter for the glutamine synthetase that was able to produce much form of phosphates – is therefore an integral part of every feeding
higher ammonium concentrations of >20 mM (Ambrosio et al., 2017). strategy (Hosseini et al., 2009; Marsh, 1984; Woern and Grossmann,
This strain was also able to sustain microalgae growth in the absence of 2023). For example, among others, monopotassium phosphate is most
any additional energy or fertilizer source. Thus, this approach can be commonly used as the main phosphorous source in many media for­
used to tailor the nitrogen excretion from diazotrophic bacteria and has mulations for fungi and bacteria cultivation (FDA, 2022; Woern and
already been shown to be able to support the growth of other microor­ Grossmann, 2023). The advantage of a bioproduction process in a closed
ganisms in a co-cultivation setting. bioreactor is that - similar to the nitrogen application - the phosphorous
does not leach into the environment during the process. Thus, a bio­
3.3.1. Challenges process allows for a targeted phosphorous application and also for a
Utilizing diazotrophic bacteria for biofertilizer production is not as potential recycling from the spent media.
straightforward as it seems. Using this production route either directly Phosphate is a finite resource because it can only be recovered by
or after mineralization to obtain fertilizer requires substantially more mining phosphate-rich rocks. These ores typically have around 25 to 35
space than ammonia production through a Haber-Bosch process. The wt% of P2O5 and are classified as sedimentary, magmatic, and guano-
first approach – intracellular production – typically results in nitrogen type deposits (van Enk et al., 2011). The main minerals in phosphate
concentrations of around 12–42 mg N L− 1 when Anabaena sp. cultures rock can be classified as francolite (fluorapatite), fluorapatite, dahllite,
are grown diazotrophically (Asmamaw et al., 2019; Barminski et al., and hydroxyapatite (van Enk et al., 2011). These minerals are estimated
2016). Typically an ammonium phosphate concentration of around 1–2 to last for the next 300–400 years, with most countries running low on
g/L (which equals around 420 mgN L− 1 using 1.5 g/L of ammonium their phosphorous reserves in the next 100 years (Cooper et al., 2011).
phosphate) is required for biomass production (e.g., fungi) (Marsh, Nevertheless, more pessimistic reports suggest that the entire global

16
L. Grossmann Biotechnology Advances 73 (2024) 108367

Table 4 especially when compared to livestock production. Phosphates in crop

Overview of phosphorous recovery approaches. Modified from (Zheng et al., production are used by the plant at an efficiency rate of around 50–70%,
2022). whereas livestock only uses around 40% of the phosphates in the pro­
Treatment Advantages Disadvantages P recovery potential vided feed with the remaining phosphates ending up in the manure
(bottlenecks) (Kebreab et al., 2012; Roberts and Johnston, 2015). Phosphate runoff is
High operational a major source of eutrophication and also although many advances have
Reliable and well-
cost on been made over the last decades it is still a problem. For example,
established Low (difficult to
Precipitation: chemicals
recover P from the agriculture is responsible for 60–80% of ‘diffuse’ P (i.e., not attributable
Chemical Difficult
chemical to any single discharge) and makes up half of the nutrient input in the
precipitation Removal efficiency recovery and
≥90–95% of P reuse of Fe/Al
precipitates) Baltic Sea together with nitrogen (Barquet et al., 2020). This runoff
precipitates possibly can be drastically reduced in a closed bioreactor system. During
Only cost- the cultivation, the microorganisms are in constant contact with the
Commercially effective with media, which minimizes the required phosphate amounts. After the
available high P
concentration
High (requires high cultivation, the media can be recycled or re-used for a new cultivation
Precipitation: P input batch (Table 1). However, it has to be acknowledged that there is limited
Required precise
Struvite High efficiency on concentration to
controls on data at the moment to understand the effects of recycling or re-using the
crystallization P recovery obtain high quality
conditions
P minerals) spent media - especially when it comes to specific nutrients such as
External source
phosphates - but some initial studies show promising results.
of Mg2+ and
NH4+ required Re-using. An early study carried out by Riese et al. (1994) re-used
Pretreatment of spent media from a mouse hybridoma cultivation that produced a
Easy operation and
low costs
the wastewater
High (needs low-
monoclonal antibody. The spent media was purified after the cultivation
may be required process in a sequential process to obtain a media that was re-used for the
cost and high-
Adsorption High selectivity
performance P same cultivation. The process was designed to remove the cells and the
and efficiency for Influence of
adsorbents) product from the media to enable the reintroduction of the purified
low concentration competing ions
P media into the process. Initially, the cells were harvested by centrifu­
Low gation. The supernatant was first concentrated by ultrafiltration before
Low operating effectiveness at
the ionic strength was reduced by an electrodialysis system, which was
costs trace level P
removal followed by another filtration step through a 200 nm membrane. The
Medium (trade-off solution was then pumped through an acidic membrane ion exchanger
Simultaneous
Biological between P recovery
treatments/
removal for Strict conditions
and sludge
to bind the antibodies and the last purification step was carried out by
organics, N, P and required using a gel filtration system. The final spent media was re-used in the
Enhanced treatment cost;
other pollutants
biological P social acceptance; same cultivation process. This resulted in a cost reduction of 52% and up
Sensitivity to
removal Low chemicals and policy and to 65% of spent media could be recycled with only slightly lower
inhibiting
(EBPR) usage environmental
substances product yields. It should be noted that this process was designed for an
concerns)
Require antibody bioproduction process, which requires high purities. Especially
sustainable
the production of algal, bacterial, or fungal biomass typically requires
management of
sewage disposal
much less purification and does not involve an isolation of a product like
Excellent an antibody. For example, a fungal bioproduction process to obtain
High capital
separation and
costs
mycoprotein biomass is often carried out using light malt extract. After
removal the fungal biomass has been removed, it should be possible to reuse the
Membrane
spent media after a purification step (Fig. 6). Studies are currently
concentrates can High energy and
be used for P operational costs High (high capital missing that describe the feasibility of this approach and how a proper
Membrane
recovery investment and nutrient composition can be ensured when the media is reused in
processes
Resource and operational cost) another cultivation process. Nonetheless, current efforts that are
energy recovery
Membrane describing how phosphate recovery can be achieved by membrane
increase the
economic
fouling filtration might be a good starting point to apply this technology to spent
challenges media.
feasibility of water
treatment Several studies have been conducted on the feasibility of using
membrane filtration approaches to recover phosphates from different
sources. Microfiltration and ultrafiltration are primarily used to
concentrate phosphorous particles or to pretreat the feed to minimize
reserve base could be exhausted by either 2072 under high-growth membrane fouling and clog the membrane. To recover phosphates,
scenarios or sometime after 2200 in zero-growth scenarios (van Enk nanofiltration, reverse osmosis, forward osmosis, and electrodialysis can
et al., 2011). At present, China and the USA produce around 50% of the be employed (Li et al., 2021). Nanofiltration and reverse osmosis are the
world's phosphorous supply and it is estimated that the resources will be techniques with the highest technology readiness level and have phos­
depleted in both countries in around 60 years if the production rates phate retention rates of >80%, depending on the feed source and the
remain at the same level (Cooper et al., 2011). Thus, new phosphorous membrane material (Li et al., 2021). Forward osmosis does not require
resources will need to be tapped in the future. While not being an im­ high-pressure pumps and has been shown to have phosphate retention
mediate threat to bioproduction it is worth starting to think about how rates of >90%. Electrodialysis is based on superimposing a voltage over
phosphates can be more efficiently used and recycled so that many more an ion-exchange membrane that promotes ion movement across the
generations have the opportunity to mine and use phosphates for pro­ membrane depending on its charge. This promotes the concentration of
ducing food, which will also minimize the effect on our landscapes and phosphorous in the anode chamber. Electrodialysis can effectively
biodiversity by minimizing new mining activities (Barquet et al., 2020). remove phosphates from liquid feeds. For example, in a model feed
Bioproduction processes that produce biomass or specific bio­ consisting of water enriched with chlorides and phosphates, the con­
polymers through precision fermentation have the potential to be more centration of phosphate was reduced from 7 to 8 mmol L− 1 to below 0.5
phosphate-efficient when compared to conventional agriculture, mmol L− 1 by electrodialysis (Zhang et al., 2013). Typically, the

17
L. Grossmann Biotechnology Advances 73 (2024) 108367

concentrated phosphate is then recovered by precipitation methods (see investigated in greater detail. Blier et al. (1995) reported that the cya­
next section). nobacteria Phormidium bohneri Schmidle was able to assimilate 69% of
On a final note, these membrane filtration approaches result in a the phosphates present in wastewater from a cheese factory. The effluent
concentrated salt solution that consists of different ions and not only was first treated using an anaerobic digestion followed by a 20% dilution
phosphates. Other ions will also be concentrated, with only electrodi­ − 1
that resulted in 16 mg P-PO3− 4 L . The purified effluent was subse­
alysis being somewhat selective because it distinguishes between cations quently treated with the phototrophic organism Phormidium bohneri
and anions (Li et al., 2021). To effectively recycle the concentrated salt Schmidle. This approach resulted in a 69% reduction in phosphates after
solution as cultivation media, the elemental composition needs to be 4 days of cultivation.
analyzed (e.g., through atomic spectroscopy) to align the composition To overcome the challenges associated with biological adsorption,
with the required media composition. Moreover, most methods authors evaluated the most economical methodologies to recover and
concentrate the phosphates in the retentate, but another approach could recycle phosphates from wastestreams. They concluded that physical
be to select a membrane pore size that allows phosphates to permeate technologies such as evaporation, membranes, or air stripping are too
but retains undesirable compounds. expensive and that other technologies such as chemical precipitation or
Recycling. Membrane filtration can be utilized to obtain a concen­ microalgae/cyanobacteria treatment are economically more feasible
trated spent media that can be re-used as a cultivation media after the (Campos et al., 2019). Chemical precipitation involves the crystalliza­
composition has been adjusted. However, the phosphates can also tion of the phosphate and is especially effective at high phosphate
directly be recycled from the spent media by different approaches: concentrations. It involves adding multivalent metal ions such as mag­
biological uptake, precipitation, and adsorption (Rittmann et al., 2011) nesium, calcium, aluminum, or ferric iron that precipitate the phos­
(Table 4). phates in the solution. The most common precipitation methods are
Biological adsorption utilizes phototrophic microorganisms such as struvite (MgNH4PO4) and calcium phosphate (e.g., Ca5(PO4)3OH)
cyanobacteria and microalgae to recover phosphates. Recycling phos­ (Rittmann et al., 2011). Struvite precipitation occurs spontaneously in
phates using microorganisms has been extensively studied and micro­ the presence of ammonium when the phosphate concentration reaches
organisms such as microalgae have been proposed as ideal organisms 100–200 mg L− 1 (Rittmann et al., 2011). Thus, this method is capable of
because of their high absorption capabilities (Solovchenko et al., 2016). recovering ammonium and phosphate at the same time. Typically,
This would, theoretically, allow growing phototrophic microorganisms magnesium chloride is mixed with appropriate amounts of a phospho­
in the spend media to assimilate nutrients and phosphates. The algae can rous salt to ensure the optimum molar ratio of magnesium, ammonium,
then be utilized as fertilizer in the main bioproduction process (see and phosphate (1:1:1) with typically slighter higher values of magne­
section 3.3 for a discussion on algae fertilizer applications). sium required to initiate the crystallization (Warmadewanthi et al.,
Microalgae are inherently adapted to low-phosphorus environments 2020). It has to be mentioned that struvite forms at alkaline pH values
and their phototrophic nature allows them to assimilate phosphates in and resolubilizes under acidic conditions (Rittmann et al., 2011).
environments without fermentable sugars. For this reason, these or­ Similarly, the highest phosphate recovery using calcium salts is achieved
ganisms possess the ability to absorb and accumulate substantial at alkaline conditions at pH >7 to form hydroxyapatite (Ca5(PO4)3OH)
amounts of phosphorus when it becomes accessible, which is commonly (Deng and Dhar, 2023). In both methods, the salts are mixed with the
referred to as ‘luxury uptake’ (Solovchenko et al., 2019). On the one phosphate-containing solution to achieve supersaturation, nucleation,
hand, this accumulation leads to devastating algae blooms, but it can growth, and agglomeration (Regy et al., 2022). The agglomerated
also be harnessed to recover phosphates from effluent, sludge, and crystals are subsequently harvested by settling methods or other liquid-
potentially from spent media to recycle it as biofertilizer. Microalgae solid separation methods such as filtration or centrifugation. Simple
store phosphate in the form of polyphosphates, which can make up to reactor types such as stirred tanks have been used to precipitate phos­
4–6% of the cell's dry mass under high phosphate feeding conditions phates, but also more sophisticated technologies such as fluidized bed
(Solovchenko et al., 2019). There have been many reports about how reactors or air-agitated column reactors have been developed (Regy
microalgae can be used to recover phosphates from municipal, indus­ et al., 2022). Thus, precipitation is a straightforward method to recover
trial, and agricultural wastewater, which have been reviewed by Solo­ phosphates from wastestreams, but studies are missing that utilize this
vchenko et al. (2019). As discussed in the previous section, phosphates method on spent media. Moreover, the low solubility of the precipitated
do not run off in a closed bioreactor system and theoretically the or­ phosphate crystals might be a major challenge for using the recovered
ganism can be cultivated until the majority of the phosphates have been phosphates again as a phosphorous source for media preparation (Ali,
assimilated before harvesting the target compound or the cells. In re­ 2007; Zhang and Ryan, 1998). The recovered phosphate crystals might
ality, however, the cultivation is typically not carried out until all be used for cultivating microorganisms that grow under more acidic
phosphates are depleted. Substantial amounts of phosphates can be conditions under which the crystals solubilize.
expected in the spent media, which could be recovered using approaches Adsorption of phosphates has also been discussed as an option to
that will be discussed below. It has to be noted that commonly an recover phosphorous, especially because it is able to recover phosphates
anaerobic digestion is performed to reduce the carbon content before the also at low concentrations, whereas the precipitation method typically
material is treated with the organism that recovers the phosphates. requires higher concentrations (Bacelo et al., 2020; Robles et al., 2020).
The high effectiveness of different microalgae and cyanobacteria has Adsorption materials derived from synthesized metal oxides and hy­
been widely recognized in many studies, which have been reviewed by droxides (such as Mg–Al layered double hydroxide or granular ferric
Mohsenpour et al. (2021). Depending on the source and the selected hydroxide), carbonate minerals (such as calcite and other calcium car­
species, P reduction in various wastestreams and wastewaters ranged bonate minerals), clay minerals (such as bentonite), zeolites (alumino­
from 13% to 99% with up to 25 mg L− 1 of total dissolved phosphates silicate minerals), porous silica, activated carbon, polymer-based
before the treatment (Mohsenpour et al., 2021). However, this approach adsorbents (such as anion exchange resins), bio-based materials (such as
comes with challenges such as associated costs, land needs, and waste scallop shells) industrial waste streams (such as fly-ash, ochre or
changing compositions that will be discussed in the next section. An steel slag) or specially designed filtration materials (such as Filtralite™)
early study used spent media from a Saccharomyces cerevisiae production have been proposed as potential compounds to bind and recover phos­
plant to grow the cyanobacteria Arthrospira platensis (Ayala and Vargas, phates (Auteri et al., 2022; Bunce et al., 2018). For example, different
1987). The spent media contained 800 ppm of total P, but only little types of anion exchange resins have been developed that effectively
detail was reported about the cultivation conditions and the recycling remove phosphates from liquid media with removal rates of 80–99%
effects of the cyanobacteria. In later studies, the effectiveness of utilizing (Bektaş et al., 2021; Bunce et al., 2018). Different types of resins have
cyanobacteria to remove phosphates from various materials was

18
L. Grossmann Biotechnology Advances 73 (2024) 108367

been developed for this purpose. The ion exchange resin relies on show that when phosphates were precipitated using the struvite
Coulomb interactions between its quaternary ammonium functional approach, the income generated accounted for only about 1/3 of the
groups (R4N+) and orthophosphates, but the main drawback is that it is required expenses of the chemical inputs alone (Mayer et al., 2016). This
not selective for phosphates (Foster and Vaneeckhaute, 2021). For this was also shown in another study that concluded: ‘The specific costs per
reason, composite adsorbents (also known as hybrid ion exchange) have kg P recovered (4 to 10 EUR/kg P) are in general higher than conven­
been developed that consist of inorganic nanoparticles and an organic tional fertilizer production (1.6 EUR/kg P)’ (Nättorp et al., 2017). The
support matrix (the resin) that are often employed in a fixed-bed column question, therefore, remains whether recovering phosphates from the
design. These are composed of, for instance, an anionic resin skeleton spent media is economically feasible. However, as mentioned, all of this
embedded with hydrated ferric oxide nanoparticles or other types of data has been collected for totally different scenarios (typically waste­
inorganic particles (Bui et al., 2018; Foster and Vaneeckhaute, 2021). water management) and actual studies on spent media are missing at the
These particles undergo ligand exchange interactions when orthophos­ moment. The cost structure might also change in the future when con­
phates are present, which allows for selective removal of phosphate ventional phosphate fertilizer becomes more expensive.
from the liquid feed (Foster and Vaneeckhaute, 2021). A last challenge is related to the composition of the recovered
The main prerequisite for materials to act as efficient adsorbents is phosphates. If they are applied again to formulate new media, it has to
the presence of positively charged functional groups that can interact be ensured that the phosphate concentration is adequate and that no
with the negatively charged phosphate ions through electrostatic in­ adverse compounds are present that may prevent the growth of the
teractions. In polymer-based adsorbents, these functional groups include target organism.
–NH+ 3 , =NH2 , ≡N , and ≡S , whereas calcium or magnesium salts have
+ + +

been used to modify the surface of biochars (Bacelo et al., 2020). Other 5. Conclusion
mechanisms that are especially relevant in metal oxides and hydroxides
include the formation of covalent bonds by a ligand exchange mecha­ Sustainable feedstocks are already available, and technologies have
nism that involves the phosphate and the metal cation (Bacelo et al., been developed to obtain sustainable fermentable sugars as well as ni­
2020). After the phosphate has been adsorbed to the adsorbent two trogen- and phosphate-containing ingredients that can be used for media
mechanisms are used to apply the recovered phosphate again as a formulation. The available technologies and challenges are summarized
nutrient. The first approach is to directly use the adsorbed phosphate in Table 1. For the three categories (fermentable sugars, N, P), ligno­
with the adsorbent without prior elution. This technique might be useful cellulosic sugars, gaseous chemoautotrophic feedstocks, green
for agricultural fertilizers where the phosphates can be released over ammonia, and phosphate recycling are the most promising technologies
time by degradation in the soil but might not be the most efficient way to to lower the emission and agricultural land burden for cellular agricul­
supply phosphates in a cultivation process. Another option is to elute the ture. However, economic constraints of fermentable sugars from ligno­
phosphate from the adsorbent and re-use the adsorbent material again cellulosic materials, gaseous chemoautotrophic feedstocks, as well as
(Bacelo et al., 2020). Elution has been shown to be effective for various electrochemical ammonia production are the main reasons why sus­
adsorbents. For example, sodium hydroxide is used in hybrid anionic tainable feedstocks have not been employed on a large scale yet.
exchange systems to replace phosphates with hydroxide ions at high pH Important solutions will include developing more efficient trans­
values (Foster and Vaneeckhaute, 2021). formation processes of lignocellulosic material into fermentable sugars.
This can be achieved through a combination of decreasing conversion
4.1. Challenges and capital costs. Also lowering the price of green hydrogen production
will have a decisive influence on the future of sustainable ammonia as
One major solution to recover phosphates from liquid media is well as on chemoautotrophic feedstocks. This will depend on developing
autotrophic algae and cyanobacteria systems that utilize the phosphates more efficient electrolyzers, having cheap water sources, and wide­
for their metabolism (Bunce et al., 2018). The algae/cyanobacteria spread economically viable renewable energy available. Overall,
grown on these phosphate-rich streams have been proposed to be used convergence research is required to solve these complex interdisci­
as a fertilizer in the agricultural system (Auteri et al., 2022). However, so plinary challenges to make sustainable cellular agriculture a reality.
far this approach has not been applied to spent media and several Beyond optimizing the media formulation, this will also include
questions remain. First, if autotrophic microorganisms are grown on continuing to improve reactor designs to meet the requirements of novel
spent media and the organism is used as a fertilizer in a cultivation media ingredients.
process, the phosphates have to be released beforehand to ensure high
phosphate availability. Second, the question remains why the autotro­ Acknowledgments
phic organism is not consumed directly instead of using it as a fertilizer.
Microalgae and cyanobacteria are known for their high nutrient density This project was supported by the Foundation for Food and Agri­
and several food-grade species are available (Grossmann et al., 2019). culture Research (FFAR) New Innovator Award 22-000115 and the
Hence, the spent media might serve as a feedstock for the cultivation of National Institute of Food and Agriculture, U.S. Department of Agri­
autotrophic organisms for direct consumption and not for the produc­ culture, and the Food Science Department at University of Massachu­
tion of cultivation fertilizers. Third, space and time requirements are setts Amherst, under project number MAS00612. The contents are solely
higher compared to other solutions such as filtration, adsorption, or the responsibility of the authors and do not necessarily represent the
precipitation. Phototrophic organisms require high surface-to-volume official views of the USDA or NIFA.
ratios, which means comparably high amounts of area would be
required to grow the organism on the spent media. The time required is
References
also higher compared to other techniques, which takes at least several
days to achieve sufficient phosphate depletion (e.g., as described above, AAF, 1980. Progress Report.
69% phosphate reduction after 4 days (Blier et al., 1995)) (Chittora Aggelopoulos, T., Bekatorou, A., Plessas, S., Koutinas, A.A., Nigam, P., 2018. Upgrading
of mixed food industry side-streams by solid-state fermentation with P. ostreatus.
et al., 2020).
Recycling 3, 12. [Link]
Another challenge is related to the cost efficiency of re-using or re- Ahlborn, J., Stephan, A., Meckel, T., Maheshwari, G., Rühl, M., Zorn, H., 2019. Upcycling
covering the phosphates from the spent media. Data is missing at pre­ of food industry side streams by basidiomycetes for production of a vegan protein
sent on whether the capital and operating expenses for the described source. Int. J. Recycl. Org. Waste Agric. 8, 447–455. [Link]
s40093-019-00317-4.
processes are economically viable. Phosphate rock is still comparably Ajmera, S., 2022. Sustainable Utilization of Fungi in Agriculture and Industry. Bentham
cheap. For example, data from wastewater treatment plants in Japan Science Publishers.

19
L. Grossmann Biotechnology Advances 73 (2024) 108367

Akermann, A., Weiermüller, J., Christmann, J., Guirande, L., Glaser, G., Knaus, A., interactions. Front. Sustain. Food Syst. 6, 915194 [Link]
Ulber, R., 2020. Brewers’ spent grain liquor as a feedstock for lactate production fsufs.2022.915194.
with Lactobacillus delbrueckii subsp. lactis. Eng. Life Sci. 20, 168–180. [Link] Bui, T.H., Hong, S.P., Yoon, J., 2018. Development of nanoscale zirconium molybdate
org/10.1002/elsc.201900143. embedded anion exchange resin for selective removal of phosphate. Water Res. 134,
Ali, M.I., 2007. Struvite crystallization in fed-batch pilot scale and description of solution 22–31. [Link]
chemistry of struvite. Chem. Eng. Res. Des. 85, 344–356. [Link] Bunce, J.T., Ndam, E., Ofiteru, I.D., Moore, A., Graham, D.W., 2018. A review of
cherd06031. phosphorus removal technologies and their applicability to small-scale domestic
Ambrosio, R., Ortiz-Marquez, J.C.F., Curatti, L., 2017. Metabolic engineering of a wastewater treatment systems. Front. Environ. Sci. 6 [Link]
diazotrophic bacterium improves ammonium release and biofertilization of plants fenvs.2018.00008.
and microalgae. Metab. Eng. 40, 59–68. [Link] Cai, J., He, Y., Yu, X., Banks, S.W., Yang, Y., Zhang, X., Yu, Y., Liu, R., Bridgwater, A.V.,
ymben.2017.01.002. 2017. Review of physicochemical properties and analytical characterization of
Amin, F.R., Khalid, H., Zhang, H., Rahman, S.U., Zhang, R., Liu, G., Chen, C., 2017. lignocellulosic biomass. Renew. Sust. Energ. Rev. 76, 309–322. [Link]
Pretreatment methods of lignocellulosic biomass for anaerobic digestion. AMB 10.1016/[Link].2017.03.072.
Express 7, 72. [Link] Calysta, 2022. First industrial-scale FeedKind® facility heralds new era of food security –
Aro, N., Ercili-Cura, D., Andberg, M., Silventoinen, P., Lille, M., Hosia, W., Nordlund, E., Calysta [WWW Document]. URL. [Link]
Landowski, C.P., 2023. Production of bovine beta-lactoglobulin and hen egg facility-heralds-new-era-of-food-security/ (accessed 1.7.24).
ovalbumin by Trichoderma reesei using precision fermentation technology and Campos, J.L., Crutchik, D., Franchi, Ó., Pavissich, J.P., Belmonte, M., Pedrouso, A.,
testing of their techno-functional properties. Food Res. Int. Ott. Ont 163, 112131. Mosquera-Corral, A., Val del Río, Á., 2019. Nitrogen and phosphorus recovery from
[Link] anaerobically pretreated agro-food wastes: a review. Front. Sustain. Food Syst. 2
Asmamaw, M., Wolde, G., Yohannes, M., Yigrem, S., Woldemeskel, E., Chala, A., Davis, J. [Link]
G., 2019. Comparison of cyanobacterial bio-fertilizer with urea on three crops and Cantera, S., Muñoz, R., Lebrero, R., López, J.C., Rodríguez, Y., García-Encina, P.A., 2018.
two soils of Ethiopia. Afr. J. Agric. Res. 14, 588–596. [Link] Technologies for the bioconversion of methane into more valuable products. Curr.
AJAR2018.13707. Opin. Biotechnol. 50, 128–135. [Link]
Auteri, N., Saiano, F., Scalenghe, R., 2022. Recycling phosphorus from agricultural Capdevila-Cortada, M., 2019. Electrifying the Haber–Bosch. Nat. Catal. 2, 1055. https://
streams: grey and green solutions. Agronomy 12, 2938. [Link] [Link]/10.1038/s41929-019-0414-4.
agronomy12122938. Cardoso Alves, S., Díaz-Ruiz, E., Lisboa, B., Sharma, M., Mussatto, S.I., Thakur, V.K.,
Ayala, F., Vargas, T., 1987. Experiments on Spirulina culture on waste-effluent media Kalaskar, D.M., Gupta, V.K., Chandel, A.K., 2023. Microbial meat: a sustainable
and at the pilot plant. In: Ragan, M.A., Bird, C.J. (Eds.), Twelfth International vegan protein source produced from agri-waste to feed the world. Food Res. Int. 166,
Seaweed Symposium, Developments in Hydrobiology. Springer Netherlands, 112596 [Link]
Dordrecht, pp. 91–93. [Link] Chehade, G., Dincer, I., 2021. Progress in green ammonia production as potential carbon-
Bacelo, H., Pintor, A.M.A., Santos, S.C.R., Boaventura, R.A.R., Botelho, C.M.S., 2020. free fuel. Fuel 299, 120845. [Link]
Performance and prospects of different adsorbents for phosphorus uptake and Chen, H., 2015. 3 - Lignocellulose biorefinery feedstock engineering. In: Chen, H. (Ed.),
recovery from water. Chem. Eng. J. 381, 122566 [Link] Lignocellulose Biorefinery Engineering. Woodhead Publishing, pp. 37–86. https://
cej.2019.122566. [Link]/10.1016/B978-0-08-100135-6.00003-X.
Balan, V., 2014. Current challenges in commercially producing biofuels from Chen, H., Dong, F., Minteer, S.D., 2020. The progress and outlook of bioelectrocatalysis
lignocellulosic biomass. ISRN Biotechnol. 2014, 1–31. [Link] for the production of chemicals, fuels and materials. Nat. Catal. 3, 225–244. https://
2014/463074. [Link]/10.1038/s41929-019-0408-2.
Banaszkiewicz, T., 2011. Nutritional value of soybean meal. In: Soybean and Nutrition. Cheng, G., Varanasi, P., Li, C., Liu, H., Melnichenko, Y.B., Simmons, B.A., Kent, M.S.,
IntechOpen. [Link] Singh, S., 2011. Transition of cellulose crystalline structure and surface morphology
Barminski, R., Storteboom, H., Davis, J.G., 2016. Development and evaluation of an of biomass as a function of ionic liquid pretreatment and its relation to enzymatic
organically certifiable growth medium for cultivation of cyanobacteria. J. Appl. hydrolysis. Biomacromolecules 12, 933–941. [Link]
Phycol. 28, 2623–2630. [Link] Cheng, M.-H., Huang, H., Dien, B.S., Singh, V., 2019. The costs of sugar production from
Barquet, K., Järnberg, L., Rosemarin, A., Macura, B., 2020. Identifying barriers and different feedstocks and processing technologies. Biofuels Bioprod. Biorefin. 13,
opportunities for a circular phosphorus economy in the Baltic Sea region. Water Res. 723–739. [Link]
171, 115433 [Link] Chittora, D., Meena, M., Barupal, T., Swapnil, P., Sharma, K., 2020. Cyanobacteria as a
Beckendorff, A., Lamp, A., Kaltschmitt, M., 2023. Optimization of hydrolysis conditions source of biofertilizers for sustainable agriculture. Biochem. Biophys. Rep. 22,
for xylans and straw hydrolysates by HPLC analysis. Biomass Convers. Biorefinery 100737 [Link]
13, 3361–3374. [Link] Chundawat, S.P.S., Uppugundla, N., Gao, D., Curran, P.G., Balan, V., Dale, B.E., 2017.
Beckinghausen, A., Odlare, M., Thorin, E., Schwede, S., 2020. From removal to recovery: Shotgun approach to increasing enzymatic Saccharification yields of ammonia fiber
an evaluation of nitrogen recovery techniques from wastewater. Appl. Energy 263, expansion pretreated cellulosic biomass. Front. Energy Res. 5 [Link]
114616. [Link] 10.3389/fenrg.2017.00009.
Bektaş, T.E., Uğurluoğlu, B.K., Tan, B., 2021. Phosphate removal by ion exchange in Clausen, E.C., Gaddy, J.L., 1993. Concentrated sulfuric acid process for converting
batch mode. Water Pract. Technol. 16, 1343–1354. [Link] lignocellulosic materials to sugars. US5188673A.
wpt.2021.072. Clausen, E.C., St, S., 1993. 54 Concentrated Sulfuric Acid.
Bello, S., Salim, I., Feijoo, G., Moreira, M.T., 2021. Inventory review and environmental Cooper, J., Lombardi, R., Boardman, D., Carliell-Marquet, C., 2011. The future
evaluation of first- and second-generation sugars through life cycle assessment. distribution and production of global phosphate rock reserves. Resour. Conserv.
Environ. Sci. Pollut. Res. 28, 27345–27361. [Link] Recycl. 57, 78–86. [Link]
12405-y. Cumberlege, T., Blenkinsopp, T., Clark, J., 2016. Assessment of Environmental Impact of
Ben-Arye, T., Levenberg, S., 2019. Tissue engineering for clean meat production. Front. FeedKind Protein. The Carbon Trust.
Sustain. Food Syst. 3 [Link] Dai, L., Reichert, C.L., Hinrichs, J., Weiss, J., 2019. Acid hydrolysis behavior of insoluble
Berlemont, R., Gerday, C., 2011. 1.18 - Extremophiles. In: Moo-Young, M. (Ed.), protein-rich fraction extracted from Chlorella protothecoides. Colloids Surf. A
Comprehensive Biotechnology, Second edition. Academic Press, Burlington, Physicochem. Eng. Asp. 569, 129–136. [Link]
pp. 229–242. [Link] colsurfa.2019.02.064.
Beswick, R.R., Oliveira, A.M., Yan, Y., 2021. Does the green hydrogen economy have a de C. L. e Penalva Santos, D., Correa, C., Amaral Alves, Y., Gomes Souza, C., A Mancebo
water problem? ACS Energy Lett. 6, 3167–3169. [Link] Boloy, R., 2023. Brazil and the world market in the development of technologies for
acsenergylett.1c01375. the production of second-generation ethanol. Alex. Eng. J. 67, 153–170. [Link]
Blanco, J., Iglesias, J., Morales, G., Melero, J.A., Moreno, J., 2020. Comparative life cycle org/10.1016/[Link].2022.09.004.
assessment of glucose production from maize starch and woody biomass residues as Deka, D.J., Gunduz, S., Kim, J., Fitzgerald, T., Shi, Y., Co, A.C., Ozkan, U.S., 2019.
a feedstock. Appl. Sci. 10, 2946. [Link] Hydrogen production from water in a solid oxide electrolysis cell: effect of Ni doping
Blier, R., Laliberté, G., de la Noüe, J., 1995. Tertiary treatment of cheese factory on lanthanum strontium ferrite perovskite cathodes. Ind. Eng. Chem. Res. 58,
anaerobic effluent with Phormidium bohneri and Micractinum pusillum. Bioresour. 22497–22505. [Link]
Technol. 52, 151–155. [Link] Deng, L., Dhar, B.R., 2023. Phosphorus recovery from wastewater via calcium phosphate
Bollmann, J., Pitchaimuthu, S., Kühnel, M.F., 2023. Challenges of industrial-scale testing precipitation: a critical review of methods, progress, and insights. Chemosphere 330,
infrastructure for green hydrogen technologies. Energies 16, 3604. [Link] 138685. [Link]
10.3390/en16083604. DoE, 2020. Integrated Strategies to Enable Lower-Cost Biofuels.
Bosque Foods, 2023. Bosque Foods. URL. [Link] (accessed DoE, 2022. U.S. ammonia prices rise in response to higher international natural gas
12.18.23). prices [WWW Document]. URL. [Link]
Brigham, C., 2019. Perspectives for the biotechnological production of biofuels from CO2 id=52358 (accessed 11.7.23).
and H2 using Ralstonia eutropha and other ‘Knallgas’ bacteria. Appl. Microbiol. Duval, S., Danyal, K., Shaw, S., Lytle, A.K., Dean, D.R., Hoffman, B.M., Antony, E.,
Biotechnol. 103, 2113–2120. [Link] Seefeldt, L.C., 2013. Electron transfer precedes ATP hydrolysis during nitrogenase
Brock, S., Kuenz, A., Prüße, U., 2019. Impact of hydrolysis methods on the utilization of catalysis. Proc. Natl. Acad. Sci. USA 110, 16414–16419. [Link]
agricultural residues as nutrient source for D-lactic acid production by pnas.1311218110.
Sporolactobacillus inulinus. Fermentation 5, 12. [Link] EC, 2012. Opinion on Furfural. Publications Office, LU.
fermentation5010012. EC, 2021. Authorisation to use certain proteins of animal origin to feed non-ruminant
Buecker, S., Grossmann, L., Loeffler, M., Leeb, E., Weiss, J., 2022. High molecular weight farmed animals.
λ-carrageenan improves the color stability of phycocyanin by associative

20
L. Grossmann Biotechnology Advances 73 (2024) 108367

Editorial, 2023. Green ammonia synthesis. Nat. Synth. 2, 581–582. [Link] Herrero, A., Muro-Pastor, A.M., Flores, E., 2001. Nitrogen control in cyanobacteria.
10.1038/s44160-023-00362-y. J. Bacteriol. 183, 411–425. [Link]
Ehlers, J.C., Feidenhans’l, A.A., Therkildsen, K.T., Larrazábal, G.O., 2023. Affordable Hickey, D.P., Lim, K., Cai, R., Patterson, A.R., Yuan, M., Sahin, S., Abdellaoui, S.,
green hydrogen from alkaline water electrolysis: key research needs from an Minteer, S.D., 2018. Pyrene hydrogel for promoting direct bioelectrochemistry: ATP-
industrial perspective. ACS Energy Lett. 8, 1502–1509. [Link] independent electroenzymatic reduction of N2. Chem. Sci. 9, 5172–5177. https://
acsenergylett.2c02897. [Link]/10.1039/C8SC01638K.
eia, 2023. United States Natural Gas Industrial Price (Dollars per Thousand Cubic Feet) Himmel, M., 2019. DOE Bioenergy Technologies Office (BETO) 2019 Project Peer Review
[WWW Document]. URL. [Link] Enzyme Engineering and Optimization WBS [Link].
(accessed 11.7.23). Holmström, S., Pitkänen, J.-P., 2021. Strains and processes for single cell protein or
Eibl, R., Senn, Y., Gubser, G., Jossen, V., van den Bos, C., Eibl, D., 2021. Cellular biomass production. WO2021084159A1.
agriculture: opportunities and challenges. Annu. Rev. Food Sci. Technol. 12, 51–73. Hoppenreijs, L.J.G., Annibal, A., Vreeke, G.J.C., Boom, R.M., Keppler, J.K., 2024. Food
[Link] proteins from yeast-based precision fermentation: simple purification of
Farias, D., de Mélo, A.H.F., da Silva, M.F., Bevilaqua, G.C., Ribeiro, D.G., Goldbeck, R., recombinant β-lactoglobulin using polyphosphate. Food Res. Int. 176, 113801
Forte, M.B.S., Maugeri-Filho, F., 2022. New biotechnological opportunities for C5 [Link]
sugars from lignocellulosic materials. Bioresour. Technol. Rep. 17, 100956 https:// Hosseini, S.M., Khosravi-Darani, K., Mohammadifar, M.A., Nikoopour, H., 2009.
[Link]/10.1016/[Link].2022.100956. Production of mycoprotein by fusarium venenatum growth on modified vogel
FDA, 2021. GRAS-Notice-AGRN-40. medium. Asian J. Chem.
FDA, 2022. GRAS Notice (GRN) No. 945. Houfani, A.A., Anders, N., Spiess, A.C., Baldrian, P., Benallaoua, S., 2020. Insights from
FeedKind, 2024. What is FeedKind? [WWW Document]. FeedKind®. URL. [Link] enzymatic degradation of cellulose and hemicellulose to fermentable sugars– a
[Link]/what-is-feedkind/ (accessed 1.7.24). review. Biomass Bioenergy 134, 105481. [Link]
Ferreira, R.G., Azzoni, A.R., Freitas, S., 2021. On the production cost of lignocellulose- biombioe.2020.105481.
degrading enzymes. Biofuels Bioprod. Biorefin. 15, 85–99. [Link] Hu, J., Zou, H., Li, F., Wei, S., Cheng, M., Dai, H., Song, T., Duan, L., 2023. Review on
bbb.2142. electrochemical reduction of nitrogen by graphdiyne-based catalysts: recent
Fialka, J., 2018. How a government program to get ethanol from plants failed. Sci. Am. advances and outlook. Energy Fuel 37, 3501–3522. [Link]
Finnigan, T.J.A., Wall, B.T., Wilde, P.J., Stephens, F.B., Taylor, S.L., Freedman, M.R., energyfuels.2c04028.
2019. Mycoprotein: the future of nutritious nonmeat protein, a symposium review. Hubalek, S., Post, M.J., Moutsatsou, P., 2022. Towards resource-efficient and cost-
Curr. Dev. Nutr. 3, nzz021. [Link] efficient cultured meat. Curr. Opin. Food Sci. 47, 100885 [Link]
[Link], 2023. A first for photosynthesis- and agriculture-free food: Protein cofs.2022.100885.
‘made from air’ officially on the menu [WWW Document]. [Link]. URL. IEA, 2022. Global Hydrogen Review 2022.
[Link] Ilić, N., Milić, M., Beluhan, S., Dimitrijević-Branković, S., 2023. Cellulases: from
ket-with-solein-ice-cream-in-singapore (accessed 1.6.24). lignocellulosic biomass to improved production. Energies 16, 3598. [Link]
Foster, X., Vaneeckhaute, C., 2021. Modifying the resin type of hybrid anion exchange 10.3390/en16083598.
nanotechnology (HAIX-Nano) to improve its regeneration and phosphate recovery IRENA, 2020. Green hydrogen cost reduction: Scaling up electrolysers to meet the 1.5C
efficiency. Npj Clean Water 4, 1–8. [Link] climate goal.
Galbe, M., Wallberg, O., 2019. Pretreatment for biorefineries: a review of common Jama, A., Widiastuti, D.P., Gafur, S., Davis, J.G., 2023. Azolla biofertilizer is an effective
methods for efficient utilisation of lignocellulosic materials. Biotechnol. Biofuels 12, replacement for urea fertilizer in vegetable crops. Sustainability 15, 6045. https://
294. [Link] [Link]/10.3390/su15076045.
Gao, Y., Cabrera Serrenho, A., 2023. Greenhouse gas emissions from nitrogen fertilizers Järviö, N., Maljanen, N.-L., Kobayashi, Y., Ryynänen, T., Tuomisto, H.L., 2021. An
could be reduced by up to one-fifth of current levels by 2050 with combined attributional life cycle assessment of microbial protein production: a case study on
interventions. Nat. Food 4, 170–178. [Link] using hydrogen-oxidizing bacteria. Sci. Total Environ. 776, 145764 [Link]
w. 10.1016/[Link].2021.145764.
Gao, L., Sun, M.H., Liu, X.Z., Che, Y.S., 2007. Effects of carbon concentration and carbon Jiao, F., Xu, B., 2019. Electrochemical ammonia synthesis and ammonia fuel cells. Adv.
to nitrogen ratio on the growth and sporulation of several biocontrol fungi. Mycol. Mater. 31, 1805173. [Link]
Res. 111, 87–92. [Link] Jones, M.D.M., Forn, I., Gadelha, C., Egan, M.J., Bass, D., Massana, R., Richards, T.A.,
García Martínez, J.B., Pearce, J.M., Throup, J., Cates, J., Lackner, M., Denkenberger, D. 2011. Discovery of novel intermediate forms redefines the fungal tree of life. Nature
C., 2022. Methane single cell protein: potential to secure a global protein supply 474, 200–203. [Link]
against catastrophic food shocks. Front. Bioeng. Biotechnol. 10 [Link] Jørgensen, H., Kristensen, J.B., Felby, C., 2007. Enzymatic conversion of lignocellulose
10.3389/fbioe.2022.906704. into fermentable sugars: challenges and opportunities. Biofuels Bioprod. Biorefin. 1,
GFI, 2023. Cultivating alternative proteins from commodity crop sidestreams. 119–134. [Link]
Gonzalez-Jordan, A., Thomar, P., Nicolai, T., Dittmer, J., 2015. The effect of pH on the Kasperski, S.D., 2023. Bioconversion of Steam-Pretreated Sugarcane Bagasse to Single-
structure and phosphate mobility of casein micelles in aqueous solution. Food Cell Protein.
Hydrocoll. 51, 88–94. [Link] Kebreab, E., Hansen, A.V., Strathe, A.B., 2012. Animal production for efficient phosphate
Grossmann, L., Weiss, J., 2021. Alternative protein sources as technofunctional food utilization: from optimized feed to high efficiency livestock. Curr. Opin. Biotechnol.
ingredients. Annu. Rev. Food Sci. Technol. 12, 93–117. [Link] 23, 872–877. [Link]
annurev-food-062520-093642. Keçebaş, A., Kayfeci, M., Bayat, M., 2019. Chapter 9 - Electrochemical hydrogen
Grossmann, L., Hinrichs, J., Weiss, J., 2019. Cultivation and downstream processing of generation. In: Calise, F., D’Accadia, M.D., Santarelli, M., Lanzini, A., Ferrero, D.
microalgae and cyanobacteria to generate protein-based technofunctional food (Eds.), Solar Hydrogen Production. Academic Press, pp. 299–317. [Link]
ingredients. Crit. Rev. Food Sci. Nutr. [Link] 10.1016/B978-0-12-814853-2.00009-6.
10408398.2019.1672137. Kobayashi, Y., Sahara, T., Ohgiya, S., Kamagata, Y., Fujimori, K.E., 2018. Systematic
Grossmann, L., Hinrichs, J., Weiss, J., 2022. Technologies for sustainable heat generation optimization of gene expression of pentose phosphate pathway enhances ethanol
in food processing. Compr. Rev. Food Sci. Food Saf. [Link] production from a glucose/xylose mixed medium in a recombinant Saccharomyces
4337.13035. cerevisiae. AMB Express 8, 139. [Link]
Haldar, D., Sen, D., Gayen, K., 2016. A review on the production of fermentable sugars Kobayashi, Y., EL-Wali, M., Guðmundsson, H., Guðmundsdóttir, E.E., Friðjónsson, Ó.H.,
from lignocellulosic biomass through conventional and enzymatic route—a Karlsson, E.N., Roitto, M., Tuomisto, H.L., 2023. Life-cycle assessment of yeast-based
comparison. Int. J. Green Energy 13, 1232–1253. [Link] single-cell protein production with oat processing side-stream. Sci. Total Environ.
15435075.2016.1181075. 873, 162318 [Link]
Hand, E., 2023. Hidden hydrogen: Earth may hold vast stores of a renewable, carbon-free Kojima, H., Nagasawa, K., Todoroki, N., Ito, Y., Matsui, T., Nakajima, R., 2023. Influence
fuel [WWW Document]. URL. [Link] of renewable energy power fluctuations on water electrolysis for green hydrogen
ydrogen-earth-may-hold-vast-stores-renewable-carbon-free-fuel (accessed 1.7.24). production. Int. J. Hydrog. Energy 48, 4572–4593. [Link]
Hann, E.C., Overa, S., Harland-Dunaway, M., Narvaez, A.F., Le, D.N., Orozco- ijhydene.2022.11.018.
Cárdenas, M.L., Jiao, F., Jinkerson, R.E., 2022. A hybrid inorganic–biological Kong-Win Chang, J., Duret, X., Berberi, V., Zahedi-Niaki, H., Lavoie, J.-M., 2018. Two-
artificial photosynthesis system for energy-efficient food production. Nat. Food 3, step thermochemical cellulose hydrolysis with partial neutralization for glucose
461–471. [Link] production. Front. Chem. 6 [Link]
Hashempour-Baltork, F., Hosseini, S.M., Assarehzadegan, M.-A., Khosravi-Darani, K., Kramer, D., 2022. Whatever happened to cellulosic ethanol? Phys. Today 75, 22–24.
Hosseini, H., 2020. Safety assays and nutritional values of mycoprotein produced by [Link]
Fusarium venenatum IR372C from date waste as substrate. J. Sci. Food Agric. 100, Krol, T., Lenz, C., 2020. Can e-fuels close the renewables power gap? A review. VGB
4433–4441. [Link] PowerTech 8.
Hauch, A., Küngas, R., Blennow, P., Hansen, A.B., Hansen, J.B., Mathiesen, B.V., Krull, S., Brock, S., Prüße, U., Kuenz, A., 2020. Hydrolyzed agricultural residues—low-
Mogensen, M.B., 2020. Recent advances in solid oxide cell technology for cost nutrient sources for l-lactic acid production. Fermentation 6, 97. [Link]
electrolysis. Science 370, eaba6118. [Link] org/10.3390/fermentation6040097.
He, S., Zou, Y., Chen, K., Jiang, S.P., 2023. A critical review of key materials and issues in Kumar, A.K., Sharma, S., 2017. Recent updates on different methods of pretreatment of
solid oxide cells. Interdiscip. Mater. 2, 111–136. [Link] lignocellulosic feedstocks: a review. Bioresour. Bioprocess. 4, 7. [Link]
idm2.12068. 10.1186/s40643-017-0137-9.
Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of Kumar, R., Singh, S., Singh, O.V., 2008. Bioconversion of lignocellulosic biomass:
lignocellulosic biomass. Bioresour. Technol. 100, 10–18. [Link] biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol. 35, 377–391.
biortech.2008.05.027. [Link]

21
L. Grossmann Biotechnology Advances 73 (2024) 108367

Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P., 2009. Methods for pretreatment of Mayer, B.K., Baker, L.A., Boyer, T.H., Drechsel, P., Gifford, M., Hanjra, M.A.,
lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Parameswaran, P., Stoltzfus, J., Westerhoff, P., Rittmann, B.E., 2016. Total value of
Chem. Res. 48, 3713–3729. [Link] phosphorus recovery. Environ. Sci. Technol. 50, 6606–6620. [Link]
Kumar, K., Mella-Herrera, R.A., Golden, J.W., 2010. Cyanobacterial heterocysts. Cold 10.1021/[Link].6b01239.
Spring Harb. Perspect. Biol. 2, a000315 [Link] Mayer, P., Ramirez, A., Pezzella, G., Winter, B., Sarathy, S.M., Gascon, J., Bardow, A.,
a000315. 2023. Blue and green ammonia production: a techno-economic and life cycle
Kumar, R., Raj, T., Næss, G., Sørensen, M., Dhawan, V., 2023. Opportunities and assessment perspective. iScience 26, 107389. [Link]
challenges in single-cell protein production using lignocellulosic material. Biofuels isci.2023.107389.
Bioprod. Biorefin. [Link] n/a. McClements, D.J., Grossmann, L., 2022. Appendix: Analysis of plant-based foods and
Kunamneni, A., Singh, S., 2005. Response surface optimization of enzymatic hydrolysis ingredients. In: McClements, D.J., Grossmann, L. (Eds.), Next-Generation Plant-
of maize starch for higher glucose production. Biochem. Eng. J. 27, 179–190. Based Foods: Design, Production, and Properties. Springer International Publishing,
[Link] Cham, pp. 535–559. [Link]
Kyriakou, V., Garagounis, I., Vourros, A., Vasileiou, E., Stoukides, M., 2020. An Minke, C., Suermann, M., Bensmann, B., Hanke-Rauschenbach, R., 2021. Is iridium
electrochemical haber-bosch process. Joule 4, 142–158. [Link] demand a potential bottleneck in the realization of large-scale PEM water
joule.2019.10.006. electrolysis? Int. J. Hydrog. Energy 46, 23581–23590. [Link]
Lagos, M.A.P., Caviativa, J.A.C., Pinzón, D.C.T., Roa, D.H.R., Basso, T.O., Lozano, M.E.V., ijhydene.2021.04.174.
2023. Xylose metabolization by a Saccharomyces cerevisiae strain isolated in Modica, V., Glávits, R., Murbach, T.S., Endres, J.R., Hirka, G., Vértesi, A., Béres, E.,
Colombia. Indian J. Microbiol. 63, 84–90. [Link] Pasics Szakonyiné, I., 2023. Toxicological evaluation of protein powder derived from
01054-z. Cupriavidus necator. J. Appl. Toxicol. JAT 43, 887–912. [Link]
Lan, R., Irvine, J.T.S., Tao, S., 2013. Synthesis of ammonia directly from air and water at jat.4432.
ambient temperature and pressure. Sci. Rep. 3, 1145. [Link] Mohsenpour, S.F., Hennige, S., Willoughby, N., Adeloye, A., Gutierrez, T., 2021.
srep01145. Integrating micro-algae into wastewater treatment: a review. Sci. Total Environ. 752,
Lau, K.Y., Pleissner, D., Lin, C.S.K., 2014. Recycling of food waste as nutrients in 142168 [Link]
Chlorella vulgaris cultivation. Bioresour. Technol. 170, 144–151. [Link] Nättorp, A., Remmen, K., Remy, C., 2017. Cost assessment of different routes for
10.1016/[Link].2014.07.096. phosphorus recovery from wastewater using data from pilot and production plants.
Lee, D.Y., Lee, S.Y., Yun, S.H., Jeong, J.W., Kim, J.H., Kim, H.W., Choi, J.S., Kim, G.-D., Water Sci. Technol. 76, 413–424. [Link]
Joo, S.T., Choi, I., Hur, S.J., 2022a. Review of the current research on fetal bovine Nguyen, L.T., Silverman, J.A., Aylen, G.I., 2023. Gas-fed fermentation reactors, systems
serum and the development of cultured meat. Food Sci. Anim. Resour. 42, 775–799. and processes utilizing gas/liquid separation vessels. US11572539B2.
[Link] Nielsen, F., Zacchi, G., Galbe, M., Wallberg, O., 2017. Sequential targeting of xylose and
Lee, B., Winter, L.R., Lee, H., Lim, D., Lim, H., Elimelech, M., 2022b. Pathways to a green glucose conversion in fed-batch simultaneous saccharification and co-fermentation
ammonia future. ACS Energy Lett. 7, 3032–3038. [Link] of steam-pretreated wheat straw for improved xylose conversion to ethanol.
acsenergylett.2c01615. Bioenergy Res. 10, 800–810. [Link]
Lee, D.Y., Yun, S.H., Lee, S.Y., Lee, Juhyun, Jr Mariano, E., Joo, S., Choi, I., Choi, J.S., Olsen, D., Jørgensen, J., Villadsen, J., Sin, G., Jørgensen, S., 2009. Modeling, Simulation
Kim, G., Lee, Jihyun, Choi, S.-H., Hur, S.J., 2023. Analysis of commercial fetal and Optimization of Single-Cell Protein Production in a U-Loop Reactor.
bovine serum (FBS) and its substitutes in the development of cultured meat. Food O’Neill, E.N., Cosenza, Z.A., Baar, K., Block, D.E., 2021. Considerations for the
Res. Int. 174, 113617 [Link] development of cost-effective cell culture media for cultivated meat production.
Lenihan, P., Orozco, A., O’Neill, E., Ahmad, M.N.M., Rooney, D.W., Walker, G.M., 2010. Compr. Rev. Food Sci. Food Saf. 20, 686–709. [Link]
Dilute acid hydrolysis of lignocellulosic biomass. Chem. Eng. J. 156, 395–403. 4337.12678.
[Link] Ortiz-Marquez, J.C.F., Do Nascimento, M., Curatti, L., 2014. Metabolic engineering of
Leung, C.C.J., Cheung, A.S.Y., Zhang, A.Y.-Z., Lam, K.F., Lin, C.S.K., 2012. Utilisation of ammonium release for nitrogen-fixing multispecies microbial cell-factories. Metab.
waste bread for fermentative succinic acid production. Biochem. Eng. J. 65, 10–15. Eng. 23, 154–164. [Link]
[Link] Osorio-Tejada, J., Tran, N.N., Hessel, V., 2022. Techno-environmental assessment of
Li, X., Shen, S., Xu, Y., Guo, T., Dai, H., Lu, X., 2021. Application of membrane separation small-scale Haber-Bosch and plasma-assisted ammonia supply chains. Sci. Total
processes in phosphorus recovery: a review. Sci. Total Environ. 767, 144346 https:// Environ. 826, 154162 [Link]
[Link]/10.1016/[Link].2020.144346. Ospina-Betancourth, C., Acharya, K., Allen, B., Entwistle, J., Head, I.M., Sanabria, J.,
Linden, J.C., Moreira, A.R., Smith, D.H., 1980. Enzymatic hydrolysis of the Curtis, T.P., 2020. Enrichment of nitrogen-fixing bacteria in a nitrogen-deficient
lignocellulosic component from vegetative forage crops. Biotechnol. Bioeng. Symp. wastewater treatment system. Environ. Sci. Technol. 54, 3539–3548. [Link]
199–212. NO.10. org/10.1021/[Link].9b05322.
Linder, T., 2023. Beyond agriculture–how microorganisms can revolutionize global food Osselin, F., Soulaine, C., Fauguerolles, C., Gaucher, E.C., Scaillet, B., Pichavant, M., 2022.
production. ACS Food Sci. Technol. 3, 1144–1152. [Link] Orange hydrogen is the new green. Nat. Geosci. 15, 765–769. [Link]
acsfoodscitech.3c00099. 10.1038/s41561-022-01043-9.
Liu, L., Wang, J., Rosenberg, D., Zhao, H., Lengyel, G., Nadel, D., 2018. Fermented Østby, H., Hansen, L.D., Horn, S.J., Eijsink, V.G.H., Várnai, A., 2020. Enzymatic
beverage and food storage in 13,000 y-old stone mortars at Raqefet cave, Israel: processing of lignocellulosic biomass: principles, recent advances and perspectives.
investigating Natufian ritual feasting. J. Archaeol. Sci. Rep. 21, 783–793. https:// J. Ind. Microbiol. Biotechnol. 47, 623–657. [Link]
[Link]/10.1016/[Link].2018.08.008. 02301-8.
Liu, Z., Shi, E., Ma, F., Jiang, K., 2021. An integrated biorefinery process for co- Padella, M., O’Connell, A., Prussi, M., 2019. What is still limiting the deployment of
production of xylose and glucose using maleic acid as efficient catalyst. Bioresour. cellulosic ethanol? Analysis of the current status of the sector. Appl. Sci. 9, 4523.
Technol. 325, 124698 [Link] [Link]
Lumpkin, R.E., 2021. Rapid pretreatment. US20210040524A1. Pajčin, I.S., Vlajkov, V., Dodić, J., Jokić, A., Grahovac, J., 2021. Biotechnological
MacFarlane, D.R., Cherepanov, P.V., Choi, J., Suryanto, B.H.R., Hodgetts, R.Y., Bakker, J. production of plant inoculants based on nitrogen-fixing bacteria. J. Process. Energy
M., Ferrero Vallana, F.M., Simonov, A.N., 2020. A roadmap to the ammonia Agric. 25, 56–63. [Link]
economy. Joule 4, 1186–1205. [Link] Pandya, R., Gandhi, P., Ji, S., Beauchamp, D., Hom, L., 2021. Compositions comprising a
Magasanik, B., 1982. Genetic Control of Nitrogen Assimilation in Bacteria. casein and methods of producing the same. US11076615B2.
Mahmud, K., Makaju, S., Ibrahim, R., Missaoui, A., 2020. Current progress in nitrogen Pankievicz, V.C.S., Irving, T.B., Maia, L.G.S., Ané, J.-M., 2019. Are we there yet? The
fixing plants and microbiome research. Plants 9, 97. [Link] long walk towards the development of efficient symbiotic associations between
plants9010097. nitrogen-fixing bacteria and non-leguminous crops. BMC Biol. 17, 99. [Link]
Makepa, D.C., Chihobo, C.H., Ruziwa, W.R., Musademba, D., 2023. A systematic review org/10.1186/s12915-019-0710-0.
of the techno-economic assessment and biomass supply chain uncertainties of Parsin, S., Kaltschmitt, M., 2024. Processing of hemicellulose in wheat straw by steaming
biofuels production from fast pyrolysis of lignocellulosic biomass. Fuel Commun. 14, and ultrafiltration – a novel approach. Bioresour. Technol. 393, 130071 [Link]
100086 [Link] org/10.1016/[Link].2023.130071.
Mankar, A.R., Pandey, A., Modak, A., Pant, K.K., 2021. Pretreatment of lignocellulosic Pattillo, A.H., 2022. Enhanced aerobic fermentation methods for producing edible fungal
biomass: a review on recent advances. Bioresour. Technol. 334, 125235 [Link] mycelium blended meats and meat analogue compositions. US20220000159A1.
org/10.1016/[Link].2021.125235. Paul, S., Bera, S., Dasgupta, R., Mondal, S., Roy, S., 2021. Review on the recent structural
Manu, M.K., Li, D., Liwen, L., Jun, Z., Varjani, S., Wong, J.W.C., 2021. A review on advances in open and closed systems for carbon capture through algae. Energy Nexus
nitrogen dynamics and mitigation strategies of food waste digestate composting. 4, 100032. [Link]
Bioresour. Technol. 334, 125032 [Link] Peplow, M., 2014. Cellulosic ethanol fights for life. Nature 507, 152–153. [Link]
Marcellin, E., Angenent, L.T., Nielsen, L.K., Molitor, B., 2022. Recycling carbon for org/10.1038/507152a.
sustainable protein production using gas fermentation. Curr. Opin. Biotechnol. 76, Peterson, E.C., Siao, R., Chua, G.G., Busran, C.T., Pavlovic, R., Thong, A., Hermansen, C.,
102723 [Link] Sofeo, N., Kanagasundaram, Y., Weingarten, M., Lindley, N., 2023. Single cell
Marsh, R.A., 1984. Fusarium graminearum. GB2137226A. protein and oil production from solid cocoa fatty acid distillates co-fed ethanol.
Massey, M.S., Davis, J.G., 2023. Beyond soil inoculation: cyanobacteria as a fertilizer Bioresour. Technol. 387, 129630 [Link]
replacement. Nitrogen 4, 253–262. [Link] Pleissner, D., Venus, J., 2014. Agricultural residues as feedstocks for lactic acid
Matsudo, M.C., Bezerra, R.P., Sato, S., Perego, P., Converti, A., Carvalho, J.C.M., 2009. fermentation. In: Green Technologies for the Environment, ACS Symposium Series.
Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as American Chemical Society, pp. 247–263. [Link]
nitrogen source. Biochem. Eng. J. 43, 52–57. [Link] ch013.
bej.2008.08.009.

22
L. Grossmann Biotechnology Advances 73 (2024) 108367

Pleissner, D., Lam, W.C., Han, W., Lau, K.Y., Cheung, L.C., Lee, M.W., Lei, H.M., Lo, K.Y., fungi. J. Agric. Food Chem. 71, 4450–4457. [Link]
Ng, W.Y., Sun, Z., Melikoglu, M., Lin, C.S.K., 2014. Fermentative jafc.2c08828.
polyhydroxybutyrate production from a novel feedstock derived from bakery waste. Sharma, M., Patel, S.N., Lata, K., Singh, U., Krishania, M., Sangwan, R.S., Singh, S.P.,
Biomed. Res. Int. 2014, e819474 [Link] 2016. A novel approach of integrated bioprocessing of cane molasses for production
Prandi, B., Faccini, A., Lambertini, F., Bencivenni, M., Jorba, M., Van Droogenbroek, B., of prebiotic and functional bioproducts. Bioresour. Technol. 219, 311–318. https://
Bruggeman, G., Schöber, J., Petrusan, J., Elst, K., Sforza, S., 2019. Food wastes from [Link]/10.1016/[Link].2016.07.131.
agrifood industry as possible sources of proteins: a detailed molecular view on the Sharp, S., 2022. How a Starch Shortage Might Impact the P&P Industry [WWW
composition of the nitrogen fraction, amino acid profile and racemisation degree of Document]. URL. [Link]
39 food waste streams. Food Chem. 286, 567–575. [Link] act-the-pp-industry (accessed 12.18.23).
foodchem.2019.01.166. Shiva Kumar, S., Himabindu, V., 2019. Hydrogen production by PEM water electrolysis –
Prévot, V., Lopez, M., Copinet, E., Duchiron, F., 2013. Comparative performance of a review. Mater. Sci. Energy Technol. 2, 442–454. [Link]
commercial and laboratory enzymatic complexes from submerged or solid-state mset.2019.03.002.
fermentation in lignocellulosic biomass hydrolysis. Bioresour. Technol. 129, Sillman, J., Nygren, L., Kahiluoto, H., Ruuskanen, V., Tamminen, A., Bajamundi, C.,
690–693. [Link] Nappa, M., Wuokko, M., Lindh, T., Vainikka, P., Pitkänen, J.-P., Ahola, J., 2019.
Pruksasri, S., Wollinger, K.K., Novalin, S., 2019. Transformation of rice bran into single- Bacterial protein for food and feed generated via renewable energy and direct air
cell protein, extracted protein, soluble and insoluble dietary fiber, and minerals. capture of CO2: can it reduce land and water use? Glob. Food Secur. 22, 25–32.
J. Sci. Food Agric. 99, 5044–5049. [Link] [Link]
Raak, N., Mangieri, N., Foschino, R., Corredig, M., 2023. Shaping future Foods through Silva, J.P.A., Carneiro, L.M., Roberto, I.C., 2013. Treatment of rice straw hemicellulosic
fermentation of side streams: microbial, chemical, and physical characteristics of hydrolysates with advanced oxidative processes: a new and promising detoxification
fermented blends from sunflower seed press cake and cheese whey. Foods 12, 4099. method to improve the bioconversion process. Biotechnol. Biofuels 6, 23. https://
[Link] [Link]/10.1186/1754-6834-6-23.
Radeloff, M.A., Beck, R.H.F., 2014. Starch hydrolysis - nutritive syrups and powders. Smith, M.D., 2019. An abbreviated historical and structural introduction to
Sugar Ind. 139, 222–227. [Link] lignocellulose. In: Understanding Lignocellulose: Synergistic Computational and
Radenkovs, V., Juhnevica-Radenkova, K., Kviesis, J., Lazdina, D., Valdovska, A., Analytic Methods, ACS Symposium Series. American Chemical Society, pp. 1–15.
Vallejo, F., Lacis, G., 2021. Lignocellulose-degrading enzymes: a biotechnology [Link]
platform for ferulic acid production from agro-industrial side streams. Foods 10, Smith, C., Hill, K., Laura, A. Torrente-Murciano, 2020. Current and future role of
3056. [Link] Haber–Bosch ammonia in a carbon-free energy landscape. Energy Environ. Sci. 13,
Raffi, M.M., Charyulu, P.B.B.N., 2021. Chapter 18 - Azospirillum-biofertilizer for 331–344. [Link]
sustainable cereal crop production: Current status. In: Viswanath, B. (Ed.), Recent Smolinka, T., 2018. Studie IndWEDe Industrialisierung der Wasser-elektrolyse in
Developments in Applied Microbiology and Biochemistry. Academic Press, -Deutschland: -Chancen und -Herausforderungen für nachhaltigen Wasserstoff für
pp. 193–209. [Link] Verkehr, Strom und -Wärme.
Regy, S., Mangin, D., Klein, J.P., Lieto, J., 2022. Phosphate Recovery by Stuvite Solovchenko, A., Verschoor, A.M., Jablonowski, N.D., Nedbal, L., 2016. Phosphorus from
Precipitation in a Stirred Reactor. wastewater to crops: an alternative path involving microalgae. Biotechnol. Adv. 34,
Reihani, S.F.S., Khosravi-Darani, K., 2018. Mycoprotein production from date waste 550–564. [Link]
using fusarium venenatum in a submerged culture. Appl. Food Biotechnol. 5, Solovchenko, A.E., Ismagulova, T.T., Lukyanov, A.A., Vasilieva, S.G., Konyukhov, I.V.,
243–352. [Link] Pogosyan, S.I., Lobakova, E.S., Gorelova, O.A., 2019. Luxury phosphorus uptake in
RethinkX, 2019. Rethinking Food and Agriculture 2020–2030. microalgae. J. Appl. Phycol. 31, 2755–2770. [Link]
Riese, U., Lütkemeyer, D., Heidemann, R., Büntemeyer, H., Lehmann, J., 1994. Re-use of 01831-8.
spent cell culture medium in pilot scale and rapid preparative purification with Soloveichik, G., 2019. Electrochemical synthesis of ammonia as a potential alternative to
membrane chromatography. J. Biotechnol. 34, 247–257. [Link] the Haber–Bosch process. Nat. Catal. 2, 377–380. [Link]
0168-1656(94)90060-4. 019-0280-0.
Risner, D., Shayevitz, A., Haapala, K., Meunier-Goddik, L., Hughes, P., 2018. Sommer, S., Hoffmann, J.L., Fraatz, M.A., Zorn, H., 2023. Upcycling of black currant
Fermentation and distillation of cheese whey: carbon dioxide-equivalent emissions pomace for the production of a fermented beverage with Wolfiporia cocos. J. Food
and water use in the production of whey spirits and white whiskey. J. Dairy Sci. 101, Sci. Technol. 60, 1313–1322. [Link]
2963–2973. [Link] Song, X., Basheer, C., Zare, R.N., 2023. Making ammonia from nitrogen and water
Rittmann, B.E., Mayer, B., Westerhoff, P., Edwards, M., 2011. Capturing the lost microdroplets. Proc. Natl. Acad. Sci. 120, e2301206120 [Link]
phosphorus. Chemosphere 84, 846–853. [Link] pnas.2301206120.
chemosphere.2011.02.001. Souza Filho, P.F., Brancoli, P., Bolton, K., Zamani, A., Taherzadeh, M.J., 2017a. Techno-
Robak, K., Balcerek, M., 2020. Current state-of-the-art in ethanol production from economic and life cycle assessment of wastewater management from potato starch
lignocellulosic feedstocks. Microbiol. Res. 240, 126534 [Link] production: present status and alternative biotreatments. Fermentation 3, 56.
micres.2020.126534. [Link]
Roberts, T.L., Johnston, A.E., 2015. Phosphorus use efficiency and management in Souza Filho, P.F., Zamani, A., Taherzadeh, M.J., 2017b. Production of edible fungi from
agriculture. Resour. Conserv. Recycl. 105, 275–281. [Link] potato protein liquor (PPL) in airlift bioreactor. Fermentation 3, 12. [Link]
resconrec.2015.09.013. 10.3390/fermentation3010012.
Robles, Á., Aguado, D., Barat, R., Borrás, L., Bouzas, A., Giménez, J.B., Martí, N., Stalker, L., Talukder, A., Strand, J., Josh, M., Faiz, M., Stalker, L., Talukder, A., Strand, J.,
Ribes, J., Ruano, M.V., Serralta, J., Ferrer, J., Seco, A., 2020. New frontiers from Josh, M., Faiz, M., 2022. Gold (hydrogen) rush: risks and uncertainties in exploring
removal to recycling of nitrogen and phosphorus from wastewater in the circular for naturally occurring hydrogen. APPEA J. 62, 361–380. [Link]
economy. Bioresour. Technol. 300, 122673 [Link] AJ21130.
biortech.2019.122673. Statista, 2023. Production Soybean Meal by Country 2022/2023 [WWW Document].
Rodrigues, M.S., Ferreira, L.S., Converti, A., Sato, S., Carvalho, J.C.M., 2010. Fed-batch Statista. URL. [Link]
cultivation of Arthrospira (Spirulina) platensis: potassium nitrate and ammonium n-worldwide-by-country/ (accessed 10.27.23).
chloride as simultaneous nitrogen sources. Bioresour. Technol. 101, 4491–4498. Sun, Z., Li, M., Qi, Q., Gao, C., Lin, C.S.K., 2014. Mixed food waste as renewable
[Link] feedstock in succinic acid fermentation. Appl. Biochem. Biotechnol. 174,
Rodríguez-Sanz, A., Fuciños, C., Torrado, A.M., Rúa, M.L., 2022. Extraction of the wheat 1822–1833. [Link]
straw hemicellulose fraction assisted by commercial endo-xylanases. Role of the Sutay Kocabaş, D., Lyne, J., Ustunol, Z., 2022. Hydrolytic enzymes in the dairy industry:
accessory enzyme activities. Ind. Crop. Prod. 179, 114655 [Link] applications, market and future perspectives. Trends Food Sci. Technol. 119,
[Link].2022.114655. 467–475. [Link]
Roser, M., 2023. Why Did Renewables Become So Cheap So Fast? Our World Data. Taha, M., Foda, M., Shahsavari, E., Aburto-Medina, A., Adetutu, E., Ball, A., 2016.
Ryu, B.-G., Kim, J., Kim, K., Choi, Y.-E., Han, J.-I., Yang, J.-W., 2013. High-cell-density Commercial feasibility of lignocellulose biodegradation: possibilities and challenges.
cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using Curr. Opin. Biotechnol. 38, 190–197. [Link]
organic waste from the brewery industry. Bioresour. Technol. Biorefine 135, copbio.2016.02.012.
357–364. [Link] Takó, M., Kerekes, E.B., Zambrano, C., Kotogán, A., Papp, T., Krisch, J., Vágvölgyi, C.,
Sadvakasova, A.K., Kossalbayev, B.D., Token, A.I., Bauenova, M.O., Wang, J., 2020. Plant phenolics and phenolic-enriched extracts as antimicrobial agents against
Zayadan, B.K., Balouch, H., Alwasel, S., Leong, Y.K., Chang, J.-S., Allakhverdiev, S.I., food-contaminating microorganisms. Antioxidants 9, 165. [Link]
2022. Influence of Mo and Fe on photosynthetic and nitrogenase activities of antiox9020165.
nitrogen-fixing cyanobacteria under nitrogen starvation. Cells 11, 904. [Link] Tanaka, M., Matsuno, R., 1985. Conversion of lignocellulosic materials to single-cell
org/10.3390/cells11050904. protein (SCP): recent developments and problems. Enzym. Microb. Technol. 7,
Sapountzi, F.M., Gracia, J.M., Weststrate, C.J., Kees-J, Fredriksson, H.O.A., 197–206. [Link]
Niemantsverdriet, J.W., 2017. Electrocatalysts for the generation of hydrogen, Tavano, O.L., Berenguer-Murcia, A., Secundo, F., Fernandez-Lafuente, R., 2018.
oxygen and synthesis gas. Prog. Energy Combust. Sci. 58, 1–35. [Link] Biotechnological applications of proteases in food technology. Compr. Rev. Food Sci.
10.1016/[Link].2016.09.001. Food Saf. 17, 412–436. [Link]
Schelling, K., 2023. Green Hydrogen to Undercut Gray Sibling by End of Decade. Tesfaw, A., 2023. The current trends of bioethanol production from cheese whey using
BloombergNEF. URL. [Link] yeasts: biological and economical perspectives. Front. Energy Res. 11 [Link]
ray-sibling-by-end-of-decade/ (accessed 1.8.24). org/10.3389/fenrg.2023.1183035.
Scholtmeijer, K., van den Broek, L.A.M., Fischer, A.R.H., van Peer, A., 2023. Potential Teter, S., 2012. DECREASE Final Technical Report Development of a Commercial Ready
protein production from lignocellulosic materials using edible mushroom forming Enzyme Application System for Ethanol.

23
L. Grossmann Biotechnology Advances 73 (2024) 108367

Tingirikari, J.M.R., Gomes, W.F., Rodrigues, S., 2017. Efficient production of prebiotic Wikandari, R., Tanugraha, D.R., Yastanto, A.J., Manikharda, Gmoser, R., Teixeira, J.A.,
gluco-oligosaccharides in orange juice using immobilized and co-immobilized 2023. Development of meat substitutes from filamentous fungi cultivated on residual
dextransucrase. Appl. Biochem. Biotechnol. 183, 1265–1281. [Link] water of tempeh factories. Molecules 28, 997. [Link]
10.1007/s12010-017-2496-2. molecules28030997.
Tokyo Gas, 2022. Synthetic Methane Could Smooth the Path to Net Zero. Witcover, J., Williams, R.B., 2020. Comparison of “advanced” biofuel cost estimates:
Tsoutsos, T., 2010. 13 - Modelling hydrolysis and fermentation processes in trends during rollout of low carbon fuel policies. Transp. Res. Part Transp. Environ.
lignocelluloses-to-bioalcohol production. In: Waldron, K. (Ed.), Bioalcohol 79, 102211 [Link]
Production, Woodhead Publishing Series in Energy. Woodhead Publishing, Woern, C., Grossmann, L., 2023. Microbial gas fermentation technology for sustainable
pp. 340–362. [Link] food protein production. Biotechnol. Adv. 108240 [Link]
Upcraft, T., Tu, W.-C., Johnson, R., Finnigan, T., Hung, N.V., Hallett, J., Guo, M., 2021. biotechadv.2023.108240.
Protein from renewable resources: mycoprotein production from agricultural Wolf, S.E., Winterhalder, F.E., Vibhu, V., de Haart, L.G.J., Guillon, O., Eichel, R.-A.,
residues. Green Chem. 23, 5150–5165. [Link] Menzler, N.H., 2023. Solid oxide electrolysis cells – current material development
Usmani, Z., Sharma, M., Awasthi, A.K., Lukk, T., Tuohy, M.G., Gong, L., Nguyen-Tri, P., and industrial application. J. Mater. Chem. A 11, 17977–18028. [Link]
Goddard, A.D., Bill, R.M., Nayak, S.C., Gupta, V.K., 2021. Lignocellulosic 10.1039/D3TA02161K.
biorefineries: the current state of challenges and strategies for efficient Wongsirichot, P., Gonzalez-Miquel, M., Winterburn, J., 2022. Recent advances in
commercialization. Renew. Sust. Energ. Rev. 148, 111258 [Link] rapeseed meal as alternative feedstock for industrial biotechnology. Biochem. Eng. J.
[Link].2021.111258. 180, 108373 [Link]
Valverde-Pérez, B., Xing, W., Zachariae, A.A., Skadborg, M.M., Kjeldgaard, A.F., Woods, P., Bustamante, H., Aguey-Zinsou, K.-F., 2022. The hydrogen economy - where is
Palomo, A., Smets, B.F., 2020. Cultivation of methanotrophic bacteria in a novel the water? Energy Nexus 7, 100123. [Link]
bubble-free membrane bioreactor for microbial protein production. Bioresour. World Bank, 2023. State and Trends of Carbon Pricing 2023.
Technol. 310, 123388 [Link] Wrobel, S., 2023. Remilk gets first regulatory permit in Israel to sell cow-free milk
Van Den Berg, L.A., Mes, J.J., Mensink, M., Wanders, A.J., 2022. Protein quality of soy [WWW Document]. URL. [Link]
and the effect of processing: a quantitative review. Front. Nutr. 9, 1004754. https:// gulatory-permit-in-israel-to-sell-cow-free-milk/ (accessed 1.13.24).
[Link]/10.3389/fnut.2022.1004754. Xu, J., Cheng, J.J., Sharma-Shivappa, R.R., Burns, J.C., 2010. Sodium hydroxide
Van Dyk, J.S., Pletschke, B.I., 2012. A review of lignocellulose bioconversion using pretreatment of switchgrass for ethanol production. Energy Fuel 24, 2113–2119.
enzymatic hydrolysis and synergistic cooperation between enzymes—factors [Link]
affecting enzymes, conversion and synergy. Biotechnol. Adv. 30, 1458–1480. Yang, J., Kim, J.E., Kim, J.K., Lee, S.H., Yu, J.-H., Kim, K.H., 2017. Evaluation of
[Link] Special issue on ACB 2011. commercial cellulase preparations for the efficient hydrolysis of hydrothermally
van Enk, R.J., van der Vee, G., Acera, L.K., Schuiling, R., Ehlert, P.A.I., 2011. The pretreated empty fruit bunches. BioResources 12, 7834–7840. [Link]
phosphate balance: Current developments and future outlook. 10.15376/biores.12.4.7834-7840.
Varnai, A., 2012. Improving Enzymatic Conversion of Lignocellulose to Platform Sugars. Yu, J., Munasinghe, P., 2018. Gas fermentation enhancement for chemolithotrophic
Voronova, A., Kim, S., Kim, D., Park, H.-Y., Hyun Jang, J., Seo, B., 2023. Systematic growth of Cupriavidus necator on carbon dioxide. Fermentation 4, 63. [Link]
degradation analysis in renewable energy-powered proton exchange membrane org/10.3390/fermentation4030063.
water electrolysis. Energy Environ. Sci. 16, 5170–5184. [Link] Yu, G., Liu, S., Feng, X., Zhang, Y., Liu, C., Liu, Y.-J., Li, B., Cui, Q., Peng, H., 2020.
D3EE01959D. Impact of ammonium sulfite-based sequential pretreatment combinations on two
Voutilainen, E., 2020. Value Chain Analysis of Sidestream-Based Feedstocks for Cellular distinct saccharifications of wheat straw. RSC Adv. 10, 17129–17142. [Link]
Agriculture. org/10.1039/d0ra01759k.
Voutilainen, E., Pihlajaniemi, V., Parviainen, T., 2021. Economic comparison of food Zhang, P., Ryan, J.A., 1998. Formation of pyromorphite in anglesite-hydroxyapatite
protein production with single-cell organisms from lignocellulose side-streams. suspensions under varying pH conditions. Environ. Sci. Technol. 32, 3318–3324.
Bioresour. Technol. Rep. 14, 100683 [Link] [Link]
Wang, L., Xia, M., Wang, H., Huang, K., Qian, C., Maravelias, C.T., Ozin, G.A., 2018. Zhang, Y., Desmidt, E., Van Looveren, A., Pinoy, L., Meesschaert, B., Van der Bruggen, B.,
Greening ammonia toward the solar ammonia refinery. Joule 2, 1055–1074. https:// 2013. Phosphate separation and recovery from wastewater by novel electrodialysis.
[Link]/10.1016/[Link].2018.04.017. Environ. Sci. Technol. 47, 5888–5895. [Link]
Wang, B., Zhang, Y., Minteer, D., 2023. Renewable electron-driven bioinorganic nitrogen Zhang, J., Choi, Y.S., Yoo, C.G., Kim, T.H., Brown, R.C., Shanks, B.H., 2015.
fixation: a superior route toward green ammonia? Energy Environ. Sci. 16, 404–420. Cellulose–hemicellulose and cellulose–lignin interactions during fast pyrolysis. ACS
[Link] Sustain. Chem. Eng. 3, 293–301. [Link]
Warmadewanthi, Rodlia, A., Ikhlas, N., Pandebesie, E.S., Bagastyo, A.Y., Herumurti, W., Zhang, B., Wu, L., Liu, X., Bao, J., 2022. Plant proteins as an alternative nitrogen source
2020. The effect of mixing rate on struvite recovery from the fertilizer industry. IOP for chiral purity L-lactic acid fermentation from lignocellulose feedstock.
Conf. Ser. Earth Environ. Sci. 506, 012013 [Link] Fermentation 8, 546. [Link]
506/1/012013. Zhang, B., Ren, D., Liu, Q., Liu, X., Bao, J., 2023. Coproduction of single cell protein and
Whittaker, J.A., Johnson, R.I., Finnigan, T.J.A., Avery, S.V., Dyer, P.S., 2020. The lipid from lignocellulose derived carbohydrates and inorganic ammonia salt with
biotechnology of quorn mycoprotein: Past, present and future challenges. In: soluble ammonia recycling. Bioresour. Technol. 384, 129345 [Link]
Nevalainen, H. (Ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges 10.1016/[Link].2023.129345.
in Biology and Biotechnology. Springer International Publishing, Cham, pp. 59–79. Zheng, Y., Wan, Y., Zhang, Y., Huang, J., Yang, Y., Tsang, D.C.W., Wang, H., Chen, H.,
[Link] Gao, B., 2022. Recovery of phosphorus from wastewater: a review based on current
Wickham, A., Davis, J.G., 2023. Optimizing organic carrot (Daucus carota var. sativus) phosphorous removal technologies. Crit. Rev. Environ. Sci. Technol. 53, 1148–1172.
yield and quality using fish emulsions, cyanobacterial fertilizer, and seaweed [Link]
extracts. Agronomy 13, 1329. [Link]

24