Applications of Humic Acids in Agriculture – A Review

Abstract:
Humic acids have a significant function in soil fertility and are thought to be
crucial for soil aggregate stabilization. They promote the soil's fertility, water
retention capacity, and nutrient uptake in plants. All of these characteristics
have resulted in a plethora of research studies on humic substances, of which
a few are discussed in this review of literature. This article might be thought of
as a condensed version of decades of research on the applications of humic
acids in agriculture, their formulation, and the various possible benefits they
may include.
Keywords: Humic acids, Agriculture, Formulation, Soil fertility, Applications of
Humic Acids
Introduction:
Humic substances (HS) are organic chemicals found in humus, the primary
organic component of soil, peat, and coal (and also a constituent of many
upland streams, dystrophic lakes, and ocean water). Humic substances were
widely regarded through the lens of acid–base theory, which classified humic
acids (HA) as organic acids and their conjugate bases, humates, as important
components of organic matter, for a long time in the 19th and 20th centuries.
Humic acids (HA) are organic acids retrieved from soil that coagulate (form
small solid pieces) when a strong-base extract is acidified, whereas fulvic
acids (FA) are organic acids that remain soluble (stay dissolved) when a
strong-base extract is acidified, according to this viewpoint. The alkali-
insoluble portion of the humus would be called humin. Humic matter in
isolation is the product of a chemical extraction from soil organic matter or
dissolved organic matter, and it represents the humic molecules that are
spread throughout the soil or water(Drosos et al., 2017; Piccolo, 2016; Source
Materials for IHSS Samples | IHSS, n.d.). As "humification" loses acceptance
as a specific instance, academic definitions of humic substances are being
debated, leading to some radical definitions that expand HS to include all
difficult-to-characterize soil organic matter at the expense of clarity. There has
also been a push to abandon the old alkali extract procedure in favor of
analyzing the soil directly, although the method's complexity limits widespread
adoption in agriculture(Lehmann & Kleber, 2015). One of the least understood
and most exciting parts of humus chemistry is the generation of humic
compounds in nature. There are three prominent ideas to explain it:
Waksman's (1932) lignin hypothesis, polyphenol theory, and Maillard's sugar-
amine condensation theory (1911)(Stevenson, 1994; Tan, 2014). These
explanations are insufficient to explain soil research findings. Humic
compounds are generated when dead plant matter, such as lignin and
charcoal, is degraded by bacteria. In the lab, humic compounds are extremely
resistant to biodegradation. The exact qualities and structure of a sample are
determined by the water or soil source as well as the extraction
circumstances. Nonetheless, the average qualities of humic compounds
synthesized in laboratories from various sources are strikingly
comparable(Mao et al., 2012; Ponomarenko & Anderson, 2011). Humic acids
are the most explored group of humic substances. These chemicals also have
industrial applications in the creation of absorbents to be utilized at metal-
poisoning sources, in addition to their importance for life. Humic substances,
which are found in humus (soil organic matter), are biomolecules that have
undergone physical, chemical, and microbiological transformations
(humification). They are significant because they are the most common
source of non-living organic material known to nature. Humic compounds
account for approximately 80% of the total carbon in terrestrial media and 60%
of the carbon dissolved in aquatic media (Peña-Méndez et al., 2005).
Role of Humic Acid in Soil:
Humic acids (HA) are organic molecules that play essential roles in improving
soil properties, plant growth, and agronomic parameters. The sources of HA
include coal, lignite, soils, and organic materials. From a detailed literature
survey, the authors highlighted that the sources of HA include coal, lignite,
soils, and organic materials. HA can positively affect soil physical, chemical,
and biological characteristics, including texture, structure, water holding
capacity, cation exchange capacity, pH, soil carbon, enzymes, nitrogen cycling,
and nutrient availability. The effect of HA on soil properties and crops is
influenced by the HA type, HA application rate, HA application mode, soil type,
solubility, molecular size, and functional group. (Ampong et al., 2022)
Furthermore, Humic acid is not a fertilizer as it does not directly provide
nutrients to plants, but is a compliment to fertilizer. A field experiment was
conducted at the experimental station of National Research center at El-Nub
aria region, Egypt, to study the role of foliar application of humic acid (as soil
conditioner 13 cm/l). The results showed that plant treated with humic acid or
nicotinamide increased significantly all morphological criteria (plant height,
leaves number, fresh and dry weights of shoots), metabolism (photosynthetic
pigment, total soluble sugar, total carbohydrates, total amino acids and
proline), mineral contents (N, P, K, Ca and Mg) and yield (grain, straw and
biology) of both cultivars amended with either recommended or half
recommended doses of NPK. Foliar application of humic acid to plant priming
in Vitamin pp induced significant increases in all studied parameters
(morphology, chemical and yield) of plants amended with recommended or
half recommended doses of NPK as compared with control plants. The
maximum yields of grain, straw and biology of both cultivars were obtained in
response to triple treatment (humic acid + nicotinamide + recommended dose
of NPK) or (humic acid + nicotinamide + half recommended dose of NPK)
respectively.(El-Bassiouny et al., 2014) In addition to this to clarify the role of
dissolved organic matter (DOM) in sorption of organic pollutants, batch
experiments on effects of two representatives of DOM (dissolved humic acid
(HA) and tannic acid (TA)) on sorption of benzotriazole (BTA) to a sandy loam
soil were conducted. Strong positive correlation between sorbed amount of
BTA and DOM confirmed the contribution of cumulative sorption by HA or TA
in enhancing BTA binding. For HA with complex structure composed of
heterogeneous fractions, its high molecular weight (>3200 Da) fraction could
be preferentially sorbed by soil, and it can enhance BTA sorption more
obviously than the low molecular weight fraction. (Wu et al., 2020) Likewise
another group of researchers interpreted the concentration and composition
of humic acids (HAs) on the biodegradation of petroleum hydrocarbons was
investigated. The HAs were obtained from oxidized brown coal by alkaline
extraction upon the mechanical treatment of coal in the presence of 3 and 8
wt. % NaOH (HA1 and HA2, respectively). It was found that solid-phase
alkaline hydrolysis in the presence of 8 wt. % NaOH with the subsequent
separation of HA2 with water led to an increase in the degree of aromaticity
and the number of phenolic groups. The soil microflora stimulated by HA1 and
HA2 had an increased destructive oil-oxidizing activity. A decrease in the
concentration of phenanthrene in solution due to the formation of a complex
upon interaction with humic acids was established by spectrophotometry.
(Linkevich et al., 2021) A study was conducted by in olive trees in order to
examine the effects in soil in application of humic acid and bio humic acid on
yield and fruit quality. they have concluded that improving yield and fruit
quality attributed to the positive effect of organic matter alone or in
combination with humic or bio-humic in increasing the cation exchange
capacity of the soil, reducing soil pH, enhancing the root development,
increasing the root/shoot ratio, and production of root hairs of olive trees
which increase the active uptake for most of the nutrients in the soil.
Therefore, bio-humic positive effects reflected on improving the yield and fruit
quality of Kalamata olive trees. (Abd El-Razek et al., 2020) In North China Plain
a study is conducted to investigate the continuous effects of humic acid on
the physicochemical properties, microbial diversity, and enzyme activities of
soil under continuous cropping peanut. As a result, humic acid increased the
yield and quality of continuous cropping peanut. To elucidate the mechanism
of humic acid affecting the soil quality, various soil quality indicators were
evaluated and compared in this study. It was found that humic acid increased
soil nutrient contents, including the total soil nitrogen, total phosphorus, total
potassium, available nitrogen, available phosphorus, available potassium, and
organic matter contents, which exhibited the maximum effect. the number of
bacteria decreased while that of fungi increased after humic acid treatment.
(Li et al., 2019) Similarly the researchers aim to determine the properties of
humic acids defining the quality of organic matter of meadow soils irrigated
for 150 years with the slope-and-flooding system. The results identified that
humic acids produced in the many-year irrigated sandy soils were identified
with a high degree of humification, which proves the relative stability of the
soil’s organic matter. It confirms the importance of meadow soils for the
carbon sequestration process. (Banach-Szott et al., 2021) There was another
experiment conducted on maize to study the role of humic acid and nitrogen in
improving its phenology, growth and the productivity of maize. Two factors
were studied using three levels of humic acid (1.5, 3 and 4.5 kg ha-1) and four
levels of nitrogen (80, 120, 160 and 200 kg ha-1). Potassium humate a product
of HA increases the release of primary macronutrients (N, P and K). Humic
acid application to the soil is strongly associated with the physical, chemical
and biological properties of the soil. Application of humic acid increased soil
acidity, soil organic carbon and cation exchange capacity. It is concluded that
the yield and yield components of Zea mays L. were maximum when applying
3 kg HA ha-1 and 160 kg N ha-1. Hence 3 kg HA ha-1 and 160 kg N ha-1 is
recommended for higher grain yield and 4.5 kg HA ha-1 and 200 kg N ha-1 for
higher crop growth. (Azeem et al., 2014) Phosphorus (P) deficiency is a major
constraint in highly weathered tropical soils. Humic substances (HS) are not
only considered an essential component of soil organic carbon (SOC), but also
well known as a bio stimulant which can perform phytohormone-like activities
to induce nutrient uptake. Under conditions of abiotic stress such as P
starvation, HS interacts not only with plant hormones signalling pathways in
secondary metabolism systems such as by increasing expression of ABA-
responsive genes and TOR transcription related to “AUX-like activity”, but also
with other pathways including those of ROS and phenylpropanoids. the
synergetic effect between HS and different types of microorganisms which
strengthens phosphorous uptake by modulating root architecture is beneficial
for P management. (Jindo et al., 2020) In the study of role of humic acid in
plant tissue culture at low nutrient level, Humus is defined as the organic
matter in soil, a mixture of partially and totally humified substances.
Leonardite is the best source for humic acid and it is extracted by using alkali
solutions like NaOH/KOH. Humic acid plays a vital role in the plant tissue
culture as growth hormone for in vitro propagation of many plant seedlings.
This practice reduces the usage of growth regulators with 2/4 and 1/4
strength MS culture media. (Dhanapal & Sekar, 2013) Plant growth-promoting
bacteria (PGPB) and humic substances (HSs) are promising options for
reducing the use of pesticides and mineral fertilizers. Research using
transcriptomic and proteomic techniques is promising to clarify how HSs and
PGPB act in plant physiology. studies can also identify bio stimulation markers
in plants; such features can assist in genetic manipulation in plant-breeding
programs seeking to increase the response of plants to bio stimulants. (Da
Silva et al., 2021) In order to study effects of humic acid foliar spraying and
nitrogen fertilizer management on yield and yield components of peanut
(Arachis hypogaea L.). increase nitrogen application up to 75 kg/ha all studied
traits were increased. In all measured traits, the treatment of 40 mg/l humic
acid foliar spraying was superior. (Moraditochaee, 2012)
Humic substances (humic and fulvic acids) are widespread, being present in
soil, water, lake sediments, peat, and different coal. A set of agrochemical
tests were conducted in order to assess the effects of the foliar fertilizer on
tomatoes, maize, sunflower, soy, sugar beet, apple, and grape vine. In the case
of foliar application of UANHUM fertilizer. Experimental data showed yield
increases ranging from 15.4% for apple to 30.6% for field tomatoes, as
compared with the unfertilized control. The results presented in this study
demonstrate that fertilizing formulations which contain humic substances
along with main nutrients (nitrogen, phosphorus and potassium), secondary
nutrients (magnesium), and micronutrients (boron) are well assimilated by
plants and have a significant contribution to yield increase and crop quality
improvement. (Carmen et al., 2018) The Humic acid (HA) is a known bio-
stimulator, but its role in salinity tolerance is not well reported. This study
examined the effect of HA (0, 250 and 500 mg L−1) on growth, water relation,
ion-flux and chlorophyll fluorescence of Urochondra setulosa treated with 0
(control), 300 (moderate salinity) and 600 (high salinity) mM NaCl. HA
application significantly promoted plant growth at all salinities. Shoot growth
of HA-treated plants increased up to 58% (FW) and 50% (DW) at 600 mM NaCl
as compared to HA untreated non-saline controls. HA application protected U.
setulosa leaf from membrane damages by decreasing hydrogen peroxide and
malondialdehyde contents, while it increased soluble sugars and proline
accumulation. HA application helped U. setulosa to get rid of toxic ions by
secreting more Na+, while retaining more K+ in the leaf via selective trans-
membrane transport. HA could be used as a natural growth stimulator to
enhance salt tolerance of plants and get higher biomass through saline
agriculture. (Bano et al., 2022) Returning straw plays an important role in
protecting the environment and maintaining the sustainable development of
agriculture. Researchers studied the effects of returning granular corn straw
on soil humus composition and humic acid structural characteristics in a
typical saline-alkali soil. The treatments were granular corn straw (GS), coarse
corn straw (CS), and control (CK). The results show that treatments GS and
CS both increased the contents of soil humus and its components in the soil
compared to CK. However, treatment GS recorded the highest significant
increase in soil humus carbon (HEC), fulvic acid carbon (FAC), and humic acid
carbon (HAC) by 17.59%, 8.32%, and 26.51%, respectively. Moreover, an
analysis of soil humic acid (HA) through an infrared spectrum, fluorescence
spectrum, and principal components analysis (PCA) showed that the
application of straw makes the structure of HA aliphatic, simple, and younger,
and also promotes the continuous renewal of humus. (Chen et al., 2020)
Production of Humic Acids:
A study was conducted to produce humic acids by submerged fermentation of
empty fruit bunch (EFB) with Trichoderma viride and to investigate the effects
of the cellulosic substrates and the organic sources of nitrogen on the
biotechnological production of these acids. The results obtained indicate the
potential application of EFB, a waste of oil palm processing, for humic acids
production. Because EFB contains cellulose, hemicellulose and lignin,
fermentations were also performed using these polymers as carbon sources,
separately or in combination. Use of either potato peptone or yeast extract as
a nitrogen source yielded nearly identical patterns of fungal growth and
production of humic acids. (Motta & Santana, 2013) The aim of this research
was to study the production of humic acids (HA) by Trichoderma reesei from
empty fruit bunches (EFBs) of palm oil processing, with a focus on the effects
of lignocellulosic content and residual lipids. EFBs from two different soils and
palm oil producers were previously characterized about their lignocellulosic
composition. Submerged fermentations were inoculated with T. reesei spores
and set up with or without residual lipids. The results showed that the soil and
the processing for removal of the palm fresh fruits were crucial to EFB quality.
Thus, EFBs were classified as type 1 (higher lignocellulosic and fatty acids
composition similar to the palm oil and palm kernel oil) and type 2 (lower
lignocellulosic content and fatty acids composition similar to palm oil). HA
production was associated with fungal growth, and it was higher without lipids
for both EFBs. The highest HA productivity was obtained from type 1 EFB
(approximately 90 mg L −1 at 48 h). Therefore, the lignocellulosic composition
and the nature of the residual lipids in EFBs play an important role in HA
production by submerged fermentation. (M. Volpi et al., 2019) Humic acid can
be produced by a Bacillus megaterium strain, AS019, using vinasse
(sugarcane mill wastewater) at an optimal concentration of 12 °Bx. Using
single factor and orthogonal experiments, the optimal culture conditions were
determined: optimum inoculum size, 15%; initial pH, 6.0; culture time, 8 days;
rotation speed, 180 rpm; and temperature, 28 °C. Under the optimal
conditions, the humic acid production rate for the strain was 49.50 g/L. The
results of characterization and infrared spectral analysis revealed that the
produced humic acid contained a variety of reactive functional groups. (Li et
al., 2018) Humic substance is vital for soil fertility and has wide applications
such as soil remediation agents, organic fertilizer and adsorbents. The results
of pH effect on artificial humic acids (AHAs) production demonstrated that
acid hydrothermal conditions are favourable for biomass waste dissolution
and formation of AHA precursors such as saccharides, furans, organic acids
and phenols; whereas alkaline hydrothermal conditions are suitable for AHAs
production from these precursors. The solid product known as hydrochar was
reduced to only 5 wt.% after this process indicating the significant
improvement of dissolution and transformation. The AHAs concentration of
28.28 ± 0.98 wt.% obtained from corn stalk was the highest yield so far, 10
times higher than ever reported. The carbon flow analysis indicated that the
selectivity of AHA-C was 43.2% of the initial carbon in corn stalk. (Shao et al.,
2022) Oxy-cracking has been developed recently as an effective technology
for converting residual feedstocks, like petroleum coke (petcoke), into valuable
commodity products. This offers a new pathway for creating valuable
products from solid waste hydrocarbon via oxygen incorporation onto the
aromatic edges, where the oxy-cracked petcoke becomes soluble in water as
oxygenated hydrocarbons. in this study, a copper-silicate (CaCuSi4O10)
material with nanocrystalline domains was introduced to enhance the
selectivity and conversion of the oxy-cracking reaction of petroleum coke. The
copper-silicate was synthesized in-house and characterized using BET, SEM,
FTIR and XRD analyses. The catalytic activity of the prepared material was
investigated by cracking the residual feedstock in an alkaline medium. The
results showed that the catalyst enables the reaction to occur at a lower
temperature with higher conversion as compared with the non-catalyzed
reaction. The oxy-cracked products were found to be in the form of humic acid
analogs with different contribution of the functionality groups such as
carboxylic, carbonyl, and sulfonic acids as confirmed by FTIR analysis.
(Manasrah et al., 2020) the influence of chemical pretreatments (nitric acid
and hydrogen peroxide) with a fungal isolate from coal on the
depolymerization of Pakistani subbituminous coal was conducted. The
chemical pretreatment exhibited enhanced production of humic acid from
13.5% to 54.2% for nitric acid and 45.7% for hydrogen peroxide. The production
of fungal-transformed liquid-derived humic acids were 36.4% and 31.1% for
nitric acid and hydrogen peroxide pretreated coal. The results showed that the
fungal-transformed humic acid has a high molecular weight, elevated
aromatic condensation with mono and poly-substituted functional groups than
the chemically treated and raw coal-derived humic acids. By combining
chemical and fungal treatment, it is possible to improve the quantity and
quality of humic acids extracted from low rank coal. (Sabar et al., 2020) South
Africa has a variety of sources for humic substances that may be utilised for
the large- scale production of specialised fertilizers. Humic substances are
dark coloured fully decomposed remains of plant and animal organic matter.
Extracting humic acids from black liquor was optimised by comparing three
possible variations to the standard humic acid-precipitation method (HAp),
namely, lignin extraction/humic acid precipitation method (LHAp), humic acid
precipitation/acid treatment method (HApA) and lignin extraction/humic acid
precipitation/acid treatment method (LHApA). The method of extraction had a
substantial impact on the quantity and quality of humic acids produced. Humic
acid precipitation method followed by humic acid precipitation/acid treatment
method (HApA) demonstrated high quantities of humic acids compared to
lignin extraction/humic acid precipitation method (LHAp) and lignin extraction/
humic acid precipitation/acid treatment method (LHApA) Analyses such as
Fourier Transform Infrared (FTIR) spectroscopy, Ultraviolet Visible
Spectroscopy (UV-VIS), High Performance Liquid Chromatography (HPLC) and
Elemental Analysis demonstrated that the quality of humic acids produced
from black liquor was similar to those reported previously. The quality of
humic acids produced was affected by both lignin extraction and acid
treatment. Humic acids from hardwood and softwood black liquor had a low
nitrogen and high sulphur content compared to those extracted from black
liquor produced from bagasse material. Although chemical and instrumental
analysis of humic acids was quite reasonable for all humic acids produced
LHAp method was favoured for the extraction of humic acids from pine liquor
due to the high quantity of humic acids produced (low S and ash contents).
The humic acids extracted by means of HAp method and HApA had a positive
effect on plant growth, when extracted from bagasse and Eucalyptus black
liquors. Humic acids extracted by HAp from pine liquor had an inhibitory effect
on plant growth. The possible process flow indicated that with the amounts of
black liquor produced from pulp industry production of humic acids in South
Africa is a possibility. HAp method was most suitable for the production of
humic acids from Eucalyptus and bagasse liquors in terms of quantity and
plant response but LHAp method was more suitable for the production of
humic acids from pine liquor in terms of both quantity and quality. (Vusie
Mema, 2006) HA is widely used in agriculture but is known to have therapeutic
properties, which are still poorly explored. Empty Fruit Bunch (EFB) is an agro-
industrial residue that is readily available at a low cost. study aimed to study
the production of HA by Trichoderma reesei in the SSF of raw fibers of EFBs
from two different palm oil producers. Profiles of HA production, cellular
protein, pH, glucose, moisture, and oxygen transfer were obtained during SSF
by EFB with and without lipids, as well as, images by electron microscopy of
fibers. Results showed efficient HA production in the raw fibers of the EFBs.
HA production followed the cellular protein (6 g HA per 100 g of fibers)
evolution of the fermentations in the absence of lipids, while the composition
of lipids greatly affected its production. The best HA production (350 mg HA
per 100 g of fibers) was obtained from EFB that was richer in lignocellulosics
and the residual lipids were similar to the fractions of palm and kernel oils,
while EFB with lower lignocellulosic presented a production 110 mg HA per
100 g of fibers. (M. P. C. Volpi et al., 2019) Empty fruit bunch (EFB), an
underutilized waste product of oil palm processing, was studied as a substrate
for the production of humic acids (HA) by a Trichoderma reesei strain by solid-
state fermentation (SSF) in Raimbault columns. Commercial HA are currently
chemically extracted from peat and coal, which are nonrenewable carbon
sources. Trichoderma sporulation and HA production are related, and the
results of this study showed that SSF stimulated fast sporulation. The
productivity related to HA was much higher than that of the biomass,
indicating an efficient utilization of EFB. These findings, added to the low cost
of EFB, make SSF an attractive process for HA production. (Motta & Santana,
2014) The effect of alkaline extraction parameters on the yield, biological
activity, and functional group composition of the humic acids (HAs) of brown
coals was investigated. It was found that the functional group composition of
the HAs can be intentionally affected by varying alkaline extraction
parameters (the amount of an alkali and the duration and temperature of the
process). It was established that the milder the conditions of alkaline
extraction, the lower the yield of HAs, but the higher their degree of
aromaticity (fa) and biological activity. (Zherebtsov et al., 2019) A study was
done in order to quantify the amount of humic acid (HA) that could be
extracted from composted pineapple leaf residue using potassium hydroxide
(KOH) produced from pineapple leaf residue, and to compare the elemental
composition, functional groups, and spectral characteristics of HA extracted
from composted pineapple leaf using 0.1 M KOH from pineapple leaf and that
of analytical grade (0.1 M KOH). The 0.1 M KOH from pineapple leaf residue
extracted 20% HA from composted pineapple leaf residue while that of the
analytical grade (0.1 M KOH) extracted 30%. The elemental composition (C, H,
N, O, and S), functional groups (carboxylic, phenolic OH, and total acidity), and
spectral characteristics of the HA extracted using the 2 extractants were
generally similar. Potassium hydroxide from pineapple leaves can be used to
extract some reasonable amount of HA without appreciably altering the
elemental and functional groups constitution as well as the spectral
characteristics. The potential of using KOH from pineapple leaf residue in HA
studies appears promising. (Ahmed et al., 2003) Researchers compares the
humic acids production of Trichoderma (viride and reesei) species using
empty fruit bunch as the substrate during submerged fermentation. The
performance of each species was compared by examining spore production in
oat medium, and the significant medium components and fermentation
conditions were identified using Plackett and Burman statistical design. For
both Trichoderma species, the results indicate that humic acids production
can be enhanced by increasing the temperature, empty fruit bunch and
peptone concentrations and by decreasing the (NH4)2SO4 concentration. T.
reesei performed better than T. viride, generating 3-fold more of humic acids.
(Fernanda & Maria, 2014) One such experiment was to determine an influence
of humic acid (HA), seaweed liquid fertilizer (SLF) and humic acid with
seaweed liquid fertilizer combination in various concentrations on the growth
of Solanum melongena. The above plant growth promoters were applied as a
foliar spray at different intervals with the following concentrations such as a
seaweed liquid fertilizer (1 to 10%), humic acid (0.1 to 1.0) and seaweed liquid
fertilizer: humic acid (7.0:0.1 to 7.1). Among the studies the maximum growth
rate was obtained at 0.4% humic acid, 7% seaweed liquid fertilizer and 7.5:
0.5% of seaweed liquid fertilizer: humic acid combination. (Prakash et al.,
2018) To examine a strategy for synthesizing humic-like acid under H₂O₂
oxidation by using biomass pretreatment liquor as a precursor and evaluate
their electron transferring capacity and metal-binding potential. The results
show that 8% H₂O₂, a temperature of 40 °C, and a reaction time of 2 h yielded
humic-like acid up to 2.9 g L−1. Compared to the commercial humic acid,
humic-like acid has a more uniform particle size and higher C (22.5%), N
(2.3%), O (33.8%), K (4.5%), and P (11.2%) contents. The hydroxyl and carboxyl
group contents were 3.3 and 2.0 times higher in humic-like acid than that in
humic acid, respectively. Moreover, humic-like acid has more electron
transferring capacity as well as metal-binding potential than commercial
humic acid, suggesting the great application potential for remediation of
heavy metal polluted soils. The reported strategy of synthesizing humic-like
acid from pretreatment liquor in this study would promote the process of
cleaner production of biorefinery industry as well as circular economy in
agriculture. (Wang et al., 2020) Rice (Oryza sativa) straw and distillation waste
of geranium (Pelargonium graveolens), two important agricultural/industrial
crop wastes were subjected to composting and vermicomposting. T. etravirine
strains GVF10 (cellulase and xylanase producing), and RVF3 (ligninase and
celluloxylanase producing) were inoculated alone or in combination. The HA
content was found to increase maximally in rice vermicompost treated with
ligninase producing strain RVF3 (85% with respect to HA standard) followed by
geranium vermicompost + RVF3. The addition of the strain GVF10 increased
HA content to about 35-62% with respect to HA standard. Results indicate that
the use of selected fungal strain(s) hold potential to produce qualitatively
superior compost and vermicompost with high HA content in a shorter period.
Use of appropriate fungal strains may increase the efficiency of composting/
vermicomposting processes producing compost and vermicompost with
higher HA content, and alleviating the problems of solid waste accumulation
and declining soil fertility. (Maji et al., 2015) The alkaline oxidation of four
Spanish coals by air was studied using a two-level factorial design with four
variables: temperature, time, alkali concentration and coal-to-alkali ratio. The
main aim was to increase the humic acid content of these coals. The best
results were obtained at the highest temperature, longest time and lowest
alkali concentration and coal-to-alkali ratio. Lower rank coals yielded the
highest humic acid concentrations after oxidation, while the subbituminous
coals were only slightly affected by treatment under the test conditions. (Juan
et al., 1990) Nitric acid is an effective material for production of humic acids. it
is an expensive item that renders the process unfavorable industrially. Study
indicated that there was a possibility to reuse the nitric acid for oxidation
however the resulting structures of humic acids was not studied. So, to
investigate the chemical and spectroscopic properties of nitrohumic acids
prepared with reused nitric acid. The coal sample was oxidized with 5%, 10%
and 15% of nitric acid and subjected to extraction for nitrohumic acids. The
results indicated that oxidation process generally improved the acidic
functional properties of the nitrohumic acids with 10% nitric acid producing the
most promising yield. The nitric acid was reused for extraction and the
percentage yield remained good at an average of 78%. Structurally, the
nitrohumic acids produced with the reused nitric acid demonstrated larger
molecular size and fewer acidic functional groups suggesting lesser extent of
structure fractionation. (Sim et al., 2007) An organic-mineral fertilizer based on
humic substances (HSs) and potassium was developed based on the alkaline
extraction of HSs from peat. The HSs have interesting properties for use as a
fertilizer since they improve the physical and chemical structure of the soil and
provide a source of organic carbon which is readily absorbable by the plants,
whereas potassium is a primary nutrient for plants. It was found that highly
decomposed peats containing a small inorganic fraction are more favorable
for the extraction of HSs. Using these peats, organic-mineral fertilizers that
meet the Brazilian legislation have been obtained for a peatextractant mixture
containing 2.57 wt.% total organic content (TOC), a K2O/TOC ratio of 1 wt.%
and an extraction time of 12 hours. (Saito & Seckler, 2014) A model humic acid
(HA) was synthesized from a strong natural antioxidant, 3,4,5-
trihydroxybenzoic acid (gallic acid-GA), in a slow oxidative polymerization/
condensation reaction catalysed by OH- at pH ca. 8. The resulting dark-brown
product (HAG), acidified to pH ca. 2, did not precipitate from the reaction
solution and it was isolated and purified by dialysis. Its physicochemical and
spectroscopic properties, as determined by means of elemental analysis, high
performance liquid chromatography (HPLC), Fourier transform infrared (FTIR),
ultravioletvisible (UV-VIS), fluorescence and electron paramagnetic resonance
(EPR) spectroscopy, showed a close resemblance to natural humic
substances. The antioxidative activity of HAG was assayed by quenching of
chemiluminescence of lucigenin and compared to that of standard
antioxidants. (Sławinska et al., 2007) Humic acid (HA) extracted from soil is
investigated in terms of its cation exchange capacity (CEC), zero point of
charge (zpc), oxygen containing functional group content, scanning electron
microscope (SEM) images, and pH. The CEC of HA that was dried was
approximately half the CEC of hydrated HA. Metal retention by HA was
studied by conducting 24 h batch equilibrium tests with lead (Pb, as PbCl2)
and cadmium (Cd, as CdCl2) and HA at pH 4 and 6, and with a mixture of HA,
mackinawite and kaolinite. The reactivity of the HA towards Pb and Cd was
reduced when it was combined with the mackinawite and kaolinite. The
mackinawite raised the pH and dissolved some of the HA, until sufficient
reactions between the metals and mixture lowered the pH enough for the
dissolved HA to solidify and remove more metal from solution. HA bonding
with the mackinawite and kaolinite reduced the HA sites available for metal
uptake. The functional group content of the HA provided indications regarding
the origin of the HA, the extent of humification and the presence of less
decomposed fractions. The CEC of the HA could conservatively predict the
amount of metal uptake. Low pH conditions are favourable for metal removal
from solution by HA because the zpc of the HA was below pH 0.5 and
because at higher pHs HA begins to dissolve. HA, especially in the hydrated
phase, may represent an important material for metal removal from waters
and wastewaters containing metals. (Coles & Yong, 2006)
Estimation of Humic Acids:
An association index based on the fluorescence anisotropy of a
diphenyloxazole probe is introduced as a quantitative parameter for the
estimation of the associations of humic acid with nonpolar organic
compounds. The variations of the index were consistent with the behaviour of
dissolved humic acids in response to solution conditions. These included salt
concentration, pH, humic acid concentration, probe concentration, and type of
humic acid. The association indices of humic acid size fractions suggest the
existence of an intermolecular component in the association process, leading
to pseudo micellar behaviour that depends on the polydispersity of the humic
acid solution. (Engebretson et al., 1996) Humic acid (HA) represents the
organic material most widespread in nature and have positive effects on plant
physiology influencing nutrient uptake and root architecture. The application
of UV/vis spectrophotometry method to HA determination after its extraction
by Sodium pyrophosphate alkali solvent. Quantitative results obtained using
this method was compared to those obtained using the CDFA and standards
methods. To examine the effect of extraction solvent volume, solutions
containing different volumes of the sodium pyrophosphate alkali solvent were
subjected to the 0.2g sample. When the volume of extraction solvent was
increased, the extraction HA concentration increased. Also, this methodology
is easier and faster than two others, which is normally used to monitor this
sort of samples. (Javanshah & Saidi, 2016) Analysis of the organic compounds
fulvic acid, humic acid, and humin in different peat and sapropel extracts from
Lithuania was performed in this study. The dominant organic compound was
bis(tert-butyldimethylsilyl) carbonate, which varied from 6.90% to 25.68% in
peat extracts. Significant amounts of acetohydroxamic, lactic, and glycolic
acid derivatives were identified in peat and sapropel extracts. Comparing the
two extraction methods, it was concluded that active maceration was more
efficient than ultrasound extraction in yielding higher amounts of organic
compounds. The highest amounts of fulvic acid (1%) and humic acid and
humin (15.3%) were determined in pure brown peat samples. (Jarukas et al.,
2021) The best recommended method was the IHSS method as giving
highest reasonable results but it is time wasting. The HPLC method is the
second recommended method with comparing to IHSS method, despite its
costs. UV- Vi’s method gives good results with just the Pow Humus, Humigan
and Mix samples. It was found that different humic acid structures resulting
from different sources, in addition NaOH solution modified the humic acid
structure. These structures have been illustrated using FTIR after IHSS
method for humic acid extraction in the fertilizer samples of Pow Humus, Mix
and Humigan. Finally, pyrophosphate solution showed higher selectivity and
milder than NaOH solution, in contrast it extracted just 30% of humic acid in
the samples and the Redox titration of this method was of high errors. (A.C.
Hollis, 1905) Humus quality was assessed by the ratio of absorbances of
pyrophosphate soil extract at the wavelengths of 400 and 600 nm (A400/
A600). Humus fractionation was performed on selected soil samples. DRIFT
spectra of individual fractions were measured. Higher pH and lower C and N
contents were found in beech forest than in spruce forest. C and N content
increases as to humus quality decreases. DRIFT spectra of fulvic acids turned
out to be unsuitable for describing differences in humus quality. DRIFT spectra
of humic acids and humin were hence more suitable. The difference between
spruce and beech forest was found in [Link]–1 (C=C bounds of benzene
rings) and 1550 cm–1 (N-H bounds in monosubstituted amides) bands
intensities. Humic acids and humin coming from the O horizons of beech
forest are relatively enriched by nitrogen functional groups. Values of humic
acids aromaticity index did not differ between beech and spruce forests.
DRIFT spectroscopy was shown as a possible method for detailed humus
quality studying. (Lenka & Marcela Rohošková and Luboš, 2018) Present work
attempts to see how different the data varies when the basic phyto-
pharmaceutical parameters performed on processed sample. Also, an
additional assay of shilajatu, to quantify fulvic acid through UV
spectrophotometry, and quantify humic acid through gravimetric method of
analysis was carried out. Lignin decomposition logic has been attempted
using UV & chromatographic assay. Chromatography was performed as per
Certified Reference Material. The data may be used as a reference parameter
for purity analysis of processed shilajatu. Results showed the shilajatu from
Nepal had high purity and greater percentage of fulvic acid compared to that
from India. (Standardization et al., 2013) In recent years, new varieties of
organic fertilizers such as vermi compost, sludge and sediment have been
added as new sources of fertilizers, which have humic acid (HA) present in it.
The researchers aim to extract and characterize the chemical compositions of
various HA using various techniques such as UV-Vis, Fourier transform
infrared spectroscopy, proton nuclear magnetic resonance (1H-NMR),
scanning electron microscopy (SEM), energy dispersive spectrometer (EDS),
atomic absorption spectrophotometer (AAS) and X-ray diffractometer (XRD).
Cow dung manure, vermi compost, sludge and lake sediment were used for
this purpose. 1H-NMR show that all HA samples were rich in aliphatic groups.
EDS analysis found HA rich in carbon and oxygen and poor in nitrogen and
presence of elements such as Na, Ca, K, Mg, Al, S, P, Cu, Fe, Cr, Co and Pb in
various proportions. It was supported by AAS analysis where elements such
as Na, K, Ca, Mg, Fe, Cu and Co were found in all HA samples in various
concentrations. SEM images and XRD analysis showed some crystal forms
found in all HA samples. These results elucidate that the chemical
composition and the molecular structure of HA samples varies as per the
origin and environment where decomposition has occurred. Present study
also documented that the spectroscopic and imaging techniques can be
successfully used to bring out the finer differences among HA extracted from
different sources. It brings out the relative merits and demerits of vermi
compost, sludge and lake sediment as compared to cow dung manure. This
analysis showed that out of the four HA samples extracted from different
sources except HA-SD can be used as fertilizer with caution to Al, Co and Cr
content.(Rupiasih & Vidyasagar, 2009) The traditional method to determine
humic content (humic and fulvic acids) in commercial fertilizers, bio
stimulants, and organic materials is based on the oxidation of the organic
carbon contained in the basic-soluble but acid-insoluble fraction (humic acids)
and the basic-acid soluble fraction (fulvic acids) of their alkaline water
extracts. This methodology, merely operational, makes it impossible to
distinguish if the quantified carbon corresponds to substances with “humic”
chemical nature or to non-humic organic matter but with similar solubility
properties to those of humic matter. The aim is to develop new methodology
that not only quantifies the humic content in commercial products (and raw
materials) but also assesses the humic quality of the quantified organic
matter. humic and fulvic (-like) fractions have been isolated/purified from
several humic and non-humic materials and characterized by means of
elemental analysis and UV-visible, fluorescence, and infrared spectroscopies,
and these data have been used to perform a discriminant analysis (DA). The
model obtained from the DA is able to discriminate humic and fulvic fractions
from apparently humic or fulvic ones and provides discriminant classification
functions that have proven to successfully predict the “humic quality” of the
fractions isolated from commercial products, after their elemental and
spectroscopic characterization. Therefore, the combination of the
fractionation, characterization, and evaluation by the DA is proposed as an
effective methodology for quantifying and assessing the quality of the humic
content claimed in the labels of commercial products. (Fuentes et al., 2018)
Study reported the determination of humic acid (HA) and sodium alginate (Na-
Alg) at glassy carbon electrode (GCE) modified with iron oxide (Fe2O3) and
copper oxide (CuO) nanoparticles (NPs). Electrocatalytic oxidation of HA and
Na-Alg at the modified electrode showed a current drop due to adsorption of
the analyte or electrode fouling effect. The limit of detection values was 5.42
μM (HA), 5.06 μM (Na-Alg) at GCE-Fe2O3 electrode and 4.88 μM (HA), 3.82
μM (Na-Alg) at GCE-CuO electrode using LSV technique. The developed
sensors can be used for detection and monitoring of HA and sodium alginate
in treated and untreated water samples. (Adekunle et al., 2022) A simple and
rapid method for the determination of humic acid and iron in solution is
described. Two absorbance measurements are required, one on an untreated
sample aliquot, and the other on an aliquot treated to enhance iron
absorptivity. Limits of detection for each component vary with concentration
of the other, but 0.01 mg 1-1 for humic acid and 0.04 μM for iron can be
achieved. For six natural waters, determinations based on independent
calibration curves for each component gave results 6-40$ higher for iron, and
6-29% higher for humic acid, than results obtained by the proposed method.
(Carpenter & Smith, 1984) A simultaneous determination method for
ammonia and humic acid in water based on the chemiluminescence method
was developed. Ammonia was separated by a porous Teflon tube (in a double
tube), and separated ammonia was reacted with hypobromite to produce
chemiluminescence. By using the synergistic enhancement effect of humic
acid for the chemiluminescence, a detection limit for ammonia of. μmol dm
was obtained without any interference from other compounds. Humic acid in
the remaining solution coming out from the double tube also reacts with
hypobromite to produce chemiluminescence. Therefore, if the ammonia
concentration is known, the humic acid concentration can be determined. The
detection limit for humic acid was. ppb. the results obtained by the present
method were consistent with those obtained by ion chromatography for
ammonia and another reported chemiluminescence method using N-
bromosuccinimide for humic acid. (DANG et al., 2021) A rapid, sensitive and
provident flow injection analysis (FIA) method was developed within the
framework of a chemometric approach for the quantification of humic acid
(HA) in the lignite obtained from Ilgın, Konya, Turkey. The proposed method
allows automatic determination of 60 samples per hour over a wide calibration
range (0–2000 mg L−1, R2: 0.9988) and needs only 10 μL of sample at a flow
rate of mobile phase (X1), 2 mL min−1; pH of mobile phase (X2), 8, and system
temperature (X3), 20 °C. It was revealed that the standard deviation (SD)
values of the proposed FIA method are lower than those of the
spectrophotometric method. (Tarhan & Kara, 2016) A high-performance liquid
chromatographic method for the determination of humic acid in
environmental samples is presented. The humic acid is chromatographed as
its sodium or calcium complex, eluting as a single, sharp peak. Coral skeletal
matter, sea water, river water, soils and plant matter were successfully
analysed. Unusual chromatographic properties such as the occurrence of
peak broadening with increased concentration appear to be due to a slow
change in the equilibrium composition of humic acid. In solution, fulvic acid
showed similar properties to humic acid. (Susic & Boto, 1989) Seven fruit-
based fertilizer samples obtained from the Ministry of Agriculture in Gaza
include (Pow Humus, Humigan, Eptic, Liquid Potasium, Liquid H.A. Organic
corrector Mix, Agriful and Agriful Antisal), have been analyzed for humic acid
percentage content using sodium pyrophosphate. The best recommended
method was the IHSS method as giving highest reasonable results but it is
time wasting. The HPLC method is the second recommended method with
comparing to IHSS method, despite its costs. UV-Vi’s method gives good
results with just the Pow Humus, Humigan and Mix samples. It was found that
different humic acid structures resulting from different sources, in addition
NaOH solution modified the humic acid structure. (Shamia et al., 2017) The
main problem of the use of N fertilizer in paddy soil Vertisols is the low
efficiency. Urea-humic acid applications at certain dosage are believed to
increase rice growth and production. A study was aimed to examine the effect
of urea and urea-humic acid to the amount and form of N-available, N uptake,
plant growth, and the production of rice. Five dosage levels of urea-humic acid
were applied for rice on Vertisols. Results of the research showed that the
urea-humic acid fertilizer could increase the content of N-NH4+ soil at 28 days
and N-NO3− soil at 42 days. The urea-humic acid could improve plant height,
number of tillers, and total dry weight. The highest rice production is achieved
by 100% humic acid-urea, especially on parameters of dry weight effectively
harvested and effectively milled, with 5.56 tons and 4.61 tons per hectare
respectively. (Suntari et al., 2015)
Characterization of Humic Acids:
Humic acid is insoluble in strong acid (pH = 1) and has an average chemical
formula C187H186O89N9S1. A low oxygen-to-carbon ratio indicates fewer acidic
functional groups than fulvic acid, the other acidic organic polymer that can be
extracted from humus. A 1:1 hydrogen-to-carbon ratio indicates a significant
degree of aromatic character (i.e., the presence of benzene rings in the
structure), whereas a high oxygen-to-carbon ratio indicates a significant
degree of aromatic character (i.e., the presence of benzene rings in the
structure). Transition and heavy metals, such as Fe3+ and Pb2+, as well as
other chemicals with aromatic or hydrophobic (water-insoluble) chemical
structures (such as organic insecticides or anthropogenic hydrocarbons), react
violently with humic acid. Its ability to adsorb various contaminants in both
terrestrial and aquatic habitats is due to this feature. (Humic Acid | Chemistry |
Britannica, n.d.). Sediment samples from six typical lakes in northeast China
revealed a link between humic acid properties and environmental
circumstances. With decreasing lake depth, the structure of humic substance
(HS), fulvic acid (FA), and humic acid (HA) tended to simplify. According to
Pearson analysis, nutritional factors and physicochemical variables dominated
the features of humic substances in deep lake sediments, whereas
physicochemical factors dominated the changes in HS in shallow lake
sediments. Nutrient and physicochemical factors accounted 64 percent and
24.2 percent of the differences in HS content in boreal deep lakes,
respectively, according to variance partitioning results. Physicochemical
factors, on the other hand, explained the majority of the variation in HS
composition (35.6%), while nutritional factors only accounted 3.4 percent of
the variation in shallow lakes. With decreasing lake depth, structural equation
models confirmed that complex components were converted into simple
components by nutritional and physicochemical variables. Changes in
environmental circumstances with lake depth, according to this study, may
lead to changes in the quality of HS in lake sediments (Song et al., 2021). By
fractionating a bulk HA (BHA) into a series of eight subsamples, each with
reasonably homogenous properties, a study was conducted to evaluate
chemical and molecular heterogeneity of HA. Elemental analysis, pyrolysis-
gas chromatography-mass spectrometry (Py-GC-MS), Fourier transform
infrared (FTIR), ultraviolet-visible (UV-vis), and solid state 13C-nuclear
magnetic resonance (13C-NMR) spectroscopy were used to characterize the
chemical and structural properties of the eight HA fractions. On HPLC
chromatographs, the results demonstrate that each HA fraction has a
relatively narrow distribution of molecular sizes, implying that ultrafiltration is
effective at fractionating widely heterogeneous humic macromolecules into
relatively homogeneous fractions. The fractions with lower molecular weights
were found to have more heterogeneous functional groups, greater O/C
atomic ratios, and higher concentrations of oxygen and lignin-derived aromatic
structure units, according to UV-vis spectroscopy and Py-GC-MS studies.
Higher molecular weight HA fractions, on the other hand, have lower oxygen
and aromatic structural unit contents, resulting in higher H/C and lower O/C
atomic ratios. As a result of this research, has generated under similar
biogeochemical settings could contain macromolecules with a variety of
chemical, structural, and molecular properties. (Li et al., 2004). Another study
found that the structural characterization of humic acid fractions was linked to
organic matter remaining on the soil surface, particularly in agricultural
systems with a variety of species. (Machado et al., 2020). This result suggests
that the chemical environment of the soil alone doesn’t affect humic acid
production but also the diverse microorganisms that may produce or may aide
in the formulation of humic acids. Spectral techniques and elemental analysis
were used to characterise and compare humic acids extracted from seven
lignite samples from six European coal basins. Although they may provide
useful information, X-ray photoelectron spectroscopy and fluorescence
spectrometry are not typically used in the study of lignite humic acids. The
chemical nature of the investigated lignite humic acids demonstrates that
they are highly similar in chemical nature, at least in terms of aromaticity,
molecular weight, and functional group distribution. The A region contains the
fluorophores of lignite humic acids, with typical maxima in the range 255–
265/460–510 nm. For humic acids and lignites, EEM spectra have the
appearance of fingerprints (as the starting material). The ability of lignite as a
parent source of humic acids for agricultural applications was evaluated
primarily based on extractability and ash content in this study (Doskočil et al.,
2018). When a group of researchers used an elemental analyzer, Fourier
transform infrared (FT-IR), and CP/MAS 13C nuclear magnetic resonance
(NMR) spectroscopy to characterize HAs, they discovered that while they
shared some chemical and physicochemical properties, they also had some
differences in the C-containing functional groups. The C/N, C/H, O/C, and O/H
ratios for the various HAs differ significantly, indicating that the elemental
makeup of the HAs from different sediments was different due to distinct
environmental conditions. (HE et al., 2008). In 2002, researchers sought to
distinguish between HAs obtained from landfill leachates and commercial
HAs in a study. The aromatic character in the leachate HS was lower than that
of commercial humic acid (Aldrich Co.), and higher in the HS of older landfill
leachate, according to the element analysis and spectrum characteristics
acquired from UV/visible, IR, and 1H and 13C NMR in the study. Humic acids
had a larger content of condensed aromatic compounds than fulvic acids from
the same sources, according to fluorescence spectra, and the spectrum of
commercial humic acid revealed that aromatic chemicals may be present in a
much more condensed and complicated form. (Kang et al., 2002). CPMAS
13C NMR spectroscopy was used to characterize humic acid (HA) extracted
from three types of composted agricultural Saudi waste of different origins,
including agricultural crop plants (P), date palm fronds (D), and animal waste
(A) in another study conducted in Saudi Arabia, where agriculture is
considered a very important sector due to their weather and climatic
conditions (A). The CPMAS spectrum evaluated the strength of each region,
suggesting that HA (P) has a lower carbohydrate content than the other two
samples. Aromatic carbon replaced by oxygen or nitrogen was more abundant
in HA (A) than in HA (D), while aliphatic compounds were more abundant in
HA (P). All of the samples had prominent peaks for the carboxyl group,
carbonyl. The carbonyl carbon, on the other hand, had no absorption in any of
them. HA (D) had the lowest carboxyl group content among the samples,
while HA (P) and HA (A) had carboxyl group values that were relatively close.
Total aliphaticity was higher than total aromaticity in all samples, with the
highest total aliphaticity in HA (P) and the lowest total aliphaticity in HA (A).
The analyzed samples' composition and functional groups did not exactly
match those of any previously published HA models, but there was some
overlap between the Dragunov's model and HAs isolated from plant PEMs. (Al-
Faiyz, 2017) A study was conducted with the goal of analyzing and comparing
the properties of humic acid and fulvic acid. Soil samples were taken at depths
of 0.00–0.10 m in a 1989 trial with four management systems (no-till, no-till
with chiseling every three years, disc harrowing, and heavy disking) and two
production systems (no-till and no-till with chiseling every three years, disc
harrowing, and heavy disking) (succession and rotation). The soil was sieved
and divided into two portions in the lab: particles with a particle size of 2 mm
or more, and particles with a particle size of less than 2 mm. An aliquot of
each sample was taken, and the humic and fulvic acids were purified for FTIR,
UV-VIS, and NMR-1H analysis. The findings revealed that the structural
characterization of the humic and fulvic acid fractions was linked to organic
matter remaining on the soil surface, particularly in agricultural systems with a
variety of species. In aromatic and aliphatic structures, aggregates less than 2
mm produce higher NMR, UV-VIS, and FTIR spectral magnitudes than those
larger than 2 mm. The aromatic and aliphatic chemical structures of
aggregates obtained from no-till soils were preserved, resulting in increased
spectral amplitude (Machado et al., 2020). In vitro gastrointestinal models
were used to examine the efficacy of encapsulating humic acid inside yeast
cell walls (YCW) to detoxify AFB1. After 10 minutes in simulated gastric fluid
(SGF) and 1 hour in simulated intestinal fluid (SIF), GMLPs from
Saccharomyces cerevisiae cell walls showed the highest AFB1 adsorption.
GMLPs are hollow porous microspheres with a diameter of 3–4 microns that
can be used to synthesise and encapsulate AFB1-absorbing nanoparticles
(NPs). By combining humic acid with ferric chloride, humic acid nanoparticles
(HA-NPs) were created within the GMLP cavity. In SIF, encapsulating HA-NPs
in GMLPs improved HA-NP stability. In SGF and SIF, the hybrid GMLP HA-NP
formulation improved AFB1 binding synergistically compared to individual
GMLP and HA components. The GMLP HA-NP-AFB1 complexes were found to
be stable in both SGF and SIF, detoxified AFB1, and were suitable for in vivo
testing when tested on a murine macrophage cell line (Hamza et al., 2019). A
recent study in Brazil was successful in identifying and distinguishing the
humic acid produced by several integrated agricultural production systems.
Crop (C), Livestock (L), Forest (F), and various combinations of these (CLFS,
LFS, and CLS) were compared to intense (INT) and extended (EXT) pastures,
as well as natural forest (NF). The HA humification index rose with soil depth,
as evidenced by more complex structures rich in semiquinone-type free
radicals (SFR) and higher fluorescence intensity, particularly in the AFS. As a
result, HA from CLFS systems showed a stronger aromaticity profile than HA
from pastures and natural forest regions, implying that carbon compounds
had a longer lifetime. These noteworthy findings are useful for creating
climate change mitigation measures and demonstrate that humic acid's
function is influenced by climate (Tadini et al., 2022). Another Brazilian
researcher used various analyzing techniques to chemically and
spectroscopically characterize humic acids (HA) isolated from bottom
sediment samples of a stream in a Brazilian subtropical micro basin (SEM).
Despite the fact that all of the samples came from the same environment, the
data revealed that the HA have distinct chemical and spectroscopic
properties, and that the location and characteristics of the sampling points
from which the sediments were collected played a significant role in the
observed differences (Giovanela et al., 2010). Chemical procedures and
spectroscopic techniques were used to characterize humic acids extracted
from six distinct Slovak soils. Humic acids from the Calcaro-Calcic Chemozem
and Gleyic Phaeozem were found to have a high degree of aromaticity, a high
content of carboxylic groups, and polycondensed aromatic and conjugated
structures, as well as low fluorescence intensity with main peaks in the long-
wavelength spectral region and very high organic free-radical concentrations.
HAs from the Dystric Planosol, Eutric Cambisol, Orthic Luvisol, and Stagno-
Gleyic Cambisol, on the other hand, were found to have a low aromaticity and
a high aliphatic character, a low degree of polycondensation, a low content of
carboxylic groups and organic free radicals, fluorescence in the intermediate-
and low-wavelength regions, and a distinct presence of amide groups and
polyssacharide structures. These differences in chemical structure and
functionalities suggest that the first two humic acids have a higher degree of
humification and chemical reactivity than the other four samples (Barančíková
et al., 1997). Humic compounds improve soil structure and fertility, as well as
nutrient uptake and root architecture, which has a good effect on plant
physiology. Only a portion of the biochemical and molecular mechanisms
behind these events are understood. Auxin has been found in HS, and a humic
compound with auxin-like action has been postulated (Trevisan et al., 2010).
There have also been studies to distinguish between resin-extracted and
alkali-extracted humic acids. The researchers combined the traditional acid-
alkali isolation approach with benzene/methanol soil pre-treatment (one alkali-
extracted fraction) and sequential resin-alkali extraction with and without
benzene/methanol soil pre-treatment (two resin- and two alkali-extracted
fractions). Low ash level and similar elemental composition were found in all
of the HAs obtained. In comparison to resin-extracted material, which is
relatively rich in carboxylic and phenolic groups, FTIR data reveals that alkali-
extracted HAs contain more aliphatics, carbohydrates, aromatics, and/or
amides. The sequential resin-alkali extraction process appears to isolate soil
HA fractions that differ in the presence of nonhumic chemicals as well as
several photophysical properties (Timofeevna Shirshova et al., 2006).
Researchers from a private biotechnology business produced a humic acid
functional fertiliser that contains 0.1 percent -5 percent micro-nano carbon, 0.1
percent -1.5 percent urease inhibitors, 8 percent -12 percent humic acid, and
the balance fertilizers by weight. Setting quantitative input control equipment
with the aid of production technological processes for producing compound
fertilizers; quantitatively adding micro-nano carbon, urease inhibitors, humic
acid, and fertilizers into processes for producing compound fertilizers with the
aid of the equipment to obtain granular fertiliser or uniformly mixed powdery
fertilizer. The humic acid functional fertiliser with the micro-nanocarbon and
the method have advantages such as overcoming the shortcomings of low
fertiliser efficiency and utilization rate of fertilizers, high greenhouse gas
emission of soil, and severe heavy metal pollution of the soil under existing
conditions with the help of the humic acid functional fertiliser and the method,
crop yield increasing and fertiliser saving can be promoted, greenhouse gas
emission can be reduced, and a reduction in greenhouse gas emissions
(ZHOUJIAN ZHANG ZHIMING Assignee, 2016). The large volume of study into
the characterization of humic acids indicates that their properties are dynamic
rather than static. They differ depending on the HA source, the environment
from which the source was taken, the pH of the soil, the soil microfauna, and a
variety of other parameters.
Formulation of Humic Acids:
Humic acids were thought to be generated in soils by the microbial
degradation of plant lignins for many decades. However, until recently, no
investigation to see if these humic acids were generated before plant debris
reached the soil was reported. As a result, there is a lack of understanding on
how to maintain or raise the humic acid content of soils for optimal fertility.
Commercial sugar cane mulch and kelp extracts were extracted with alkali
and humic acid concentration was determined. Fluorescence
spectrophotometry was used to positively identify humic acids in the extracts,
demonstrating that humic acids are generated in senescent plant and algal
matter before reaching the soil, where they are tightly bonded to the soil and
resistant to microbial decomposition. Wind and water erosion, as well as
water leaching, remove humic acids from soils, therefore they must be
replaced on a regular basis. This study reveals that humic acid can be restored
or reinforced in soils simply by recycling plant and algal matter, or by adding
decomposed plant or algal matter from outside sources such as composts,
mulch, peat, and lignite coals(Susic, 2016). It was also found that over
standard composting, hyperthermophilic pretreatment composting (HPC)
provides the advantages of increased composting efficiency and accelerated
humic substance (HS) (CC). During the HPC of pig manure, the researchers
looked at the degree of humification and the microbial community succession.
HS content increased by 13.72 percent in HPC and 29.93 percent in sterilised
HPC inoculated with 1 percent CC (HPC I) during composting, compared to
8.76 percent in CC and 7.12 percent in sterilised CC inoculated with 1 percent
HPC during composting (CC I). In comparison to CC and CC I, HPC and HPC I
have higher levels of HA-like and fulvic acid-like components. The results
demonstrated that physicochemical variables, particularly pH, were important
in accelerating humification in HPC, and that both physicochemical properties
and the microbial community contributed to the synthesis of HA (Cao et al.,
2022). Nowadays, the general consensus on the matter is that it accumulates
over time as a byproduct of microbial metabolism. (Humic Acid | Chemistry |
Britannica, n.d.) Researchers have identified a strategy to formulate humic-like
acid under H2O2 oxidation by using biomass pretreatment liquor as a
precursor and have then evaluated their electron transferring capacity and
metal-binding potential. The results yielded show that the humic-like acid
produced by their method has more electron transferring capacity as well as
metal-binding potential than commercial humic acid, suggesting the great
application potential for remediation of heavy metal polluted soils.
(Wang1985, n.d.). A study conducted in 2016 has also determined the
influence of vermiwash and humic acid and their various combinations and
concentrations on the growth of Allium cepa. Vermiwash and humic acid and
combinations of vermiwash and humic acid at a combination of 3:1.5% was
found to be the most effective formulation. (Prakash et al., 2016). Various
other environmental and biological factors either have an effect on the
formulation of humic acid or increase its potency in the soil. To replace
chemical fertilizers, a bio formulation containing humic acid and an
appropriate microbe, Pseudomonas fluorescens, has been developed. This
liquid formulation is easy to use with irrigation and has a long shelf life, zero
contamination, no need for carriers, convenience of handling, storage, and
transportation. The combined formulation of humic acid and the bacterium
Pseudomonas fluorescens can be employed for crop protection as well as
increased production. (Pushpa et al., 2014) A study was conducted to find the
effects of biochar amendment of compost pits to increase the amount of
humic acid synthesized. The content of humic acid carbon in the compost pit
amended with biochar was found to be 16.9% more than that of the control.
(Wang et al., 2014) To increase its effectiveness or to get added advantages,
humic acids are also synthesized based on other metal ions. A simple
technique was used to develop an affordable and multifunctional Cu (II)-based
water-dispersible humic acid (Cu-WH) fungicide with growth-promoting
capabilities. A green approach was used to successfully synthesis a 3D
molybdate carbon hierarchical nanosphere (MoO2-C-HN) catalyst, which was
used in the solid-phase activation of lignite to produce water-dispersible humic
acid. A simple reaction between Cu (II) and the humic acid resulted in Cu (II)-
based water-dispersible humic acid (Cu-WH). In laboratory incubation tests,
the resulting Cu-WH showed good antifungal activity against Rhizoctonia
solani. (Y. Tang et al., 2019) A co-precipitation approach utilizing ammonium
hydroxide was used to coat magnetite nanoparticles (Fe3O4) onto humic
acids (HA). The HA was successfully coated onto the surface of Fe3O4 after
being collected from peat soil in Sambutan, East Kalimantan, Indonesia.
(Koesnarpadi et al., 2015) Another group of researchers used a chemical co-
precipitation approach to make humic acid (HA)-coated Fe 3O 4 (Fe 3O 4/HA)
superparamagnetic nanoparticles using inexpensive and environmentally safe
iron salts and HA. In neutral water, the Fe3O 4/HA nanoparticles had a faster
adsorption rate and a better removal capacity of the cationic organic dye
Methylene blue (MB). Furthermore, the MB desorption could be finished
quickly. The Fe 3O 4/HA nanoparticles' reused performance was likewise
shown to be outstanding. (Zhou et al., 2011). To make HA/ZnO nanoparticles,
a group of researchers employed a simple one-pot approach incorporating
zinc oxide nanoparticles (ZnO) cores (20-35 nm in diameter) and humic acid
shells. The effect of HA/ZnO NPs on the germination profile of Brassica
campestris was investigated. They found that exposing plants to HA/ZnO NPs
boosted germination by 83 percent, root length by 60 percent, shoot length by
78 percent, fresh weight by 71.4 percent, and dry weight by 71.4 percent (45.9
percent). The HA/ZnO NPs were used as a co-fertilizer to improve the
germination of Brassica campestris seeds. The researchers also discovered
that the seeds may have assimilated NPs, which may have triggered the union
of certain catalysts that function with food activation. Their research revealed
that HA/ZnO NPs can be a useful co-compost, providing zinc. (Pirzada et al.,
2022) Although the methods for the synthesis of humic acids are complex,
one of the most important pathways is the oxidative polymerization of
polyphenols (Kononova 1966; Stevenson 1982). Enzymes (e.g., Haider et al.
1975; Flaig et al. 1975) and inorganic components can both speed up oxidative
polymerization (e.g., Kyuma and Kawaguchi 1964; Shindo and Huang 1982;
Kumada 1987). Shindo and Huang (1985) and Shindo and Higashi (1986)
compared the relative effectiveness of various oxides, primary minerals, and
clay minerals in promoting the synthesis of humic polymers from
hydroquinone, and discovered that Mn (IV) oxide had the most striking
promoting effect among the inorganic components studied. (Shindo, 1990) In
order to boost the efficacy of humic acid, a researcher employed graft
copolymerization of humic acid (HA) with styrene to make humic acid graft
copolymer (PSt-g-HA). PSt-g-HA acid and untreated humic were used to test
the sorption of three different organic pesticides: parathion-methyl, carbaryl,
and carbofuran. Because the grafted copolymerization of polystyrene went
into condensed domains in HA, while the polar function groups, such as
carboxyl and phenolic hydroxyl group, were removed, the PSt-g-HA had a
comparatively high aromatic carbon content. The sorption capacity of three
organic pesticides (parathion-methyl, carbaryl, and carbofuran) on PSt-g-HA
rose by 64.1 percent to 95.2 percent due to the aforementioned factors. (C.
Yang et al., 2014) Even if fertilizers and other chemicals aid in enhancing
agricultural production, the use of chemical fertilizers and pesticides on a
regular basis will cause soil fertility to deteriorate over time. The only way to
restore soils is to use agriculturally significant microbes in various
combinations. Researchers have devised a bio formulation that uses humic
acid and a suitable microbe called Pseudomonas fluorescens to replace
chemical fertilizers. This liquid formulation is easy to use with irrigation and
has a long shelf life, zero contamination, no need for carriers, convenience of
handling, storage, and transportation. The combined formulation of humic
acid and the bacterium Pseudomonas fluorescens can be employed for crop
protection as well as increased production. The survivability of the liquid bio-
formulation as well as its inhibitory properties against Fusarium oxysporum, a
fungus that causes tomato wilt, were examined and compared. Two crop
kinds, radish and tomato, were studied in the field. The bio-formulations were
tested for cell viability using the plate count method. When compared to the
control, the formulation containing humic acid exhibited higher support for
viable cells as well as leafing characteristics in pots stored in the field in both
vivo and in vitro. The mixture was able to prevent Fusarium-induced tomato
wilt, proving its protective ability. (Pushpa et al., 2014) Conservation
agriculture (CA) has been found to improve soil organic carbon storage in a
variety of soil types and cropping systems by researchers all over the world.
However, research on the influence of CA on soil humic acid concentration,
stability, and availability of functional groups is limited. In an Alfisols, an
experiment was carried out with four scenarios (designated as Sc), namely
conventional farmers practise (Sc1) of transplanted rice (TPR) followed by
conventional tilled broadcasted wheat (CT- wheat) with residue removal,
transplanted rice (TPR) followed by zero tillage (ZT) wheat and mung bean
with partial residue retention (Sc2), direct seeded rice (DSR) followed by ZT-
wheat and mung bean at the research farm of ICAR-Central Soil Salinity
Research Institute, Karnal, Haryana, India. The goal of this research was to
determine the humic acid (HA) content and stability of soil carbon using a
desorption study, as well as to describe the functional groups of HAS using
Fourier Transform Infrared (FTIR) spectroscopy under various situations.
Following nine years of research, HA was recovered from soil samples
collected from 0 to 15 and 15–30 cm depths after rice harvesting in 2018. Sc4
had the highest HA concentration at 0–15 cm soil depth (4.23 g/kg) and 15–
30 cm soil depth (2.08 g/kg), according to the findings. As a result, CA is
effective not only in enriching organic carbon in soil, but also in forming a more
permanent HA complex with soil particles, which has long-term consequences
in soil carbon sequestration for future generations (Datta et al., 2022). Humic
substances (HS) are a sign of fertile soils; however, soils are losing humic
matter at an increasing rate. This is primarily due to over-cultivation caused by
humans. Artificial humic acid (A-HA) and artificial fulvic acid were synthesized
from agricultural litter, and their properties were shown to be quite similar to
those of natural HS isolated from soil. These samples were mixed into black
soils, and the activity and availability of nutrients were measured. The findings
show that the amount of dissolved organic matter (DOM) and total organic
carbon (TOC) in the water has significantly increased (C. Tang et al., 2021).
Humic acid was recovered from an aqueous solution of sodium humate using
polymer inclusion membranes in an attempt to characterise it. Treatment of
lignite from Turkey's Kahramanmaraş Karimnagar’s region yielded sodium
humate. Lignite can be used as a source of humic acid in organic fertilisers.
PIMs were made with cellulose triacetate as the polymer,
tricaprylmethylammonium chloride (Aliquat-336) as the cationic carrier,
modifier tributyl phosphate, and plasticizer 2-nitrophenyl pentyl ether as the
plasticizer. The separation of fulvic and humic acid from sodium humate was
accomplished using a permeation cell (Manzak et al., 2017). Another study
was undertaken to synthesize artificial humic acid (A-HA) utilizing biological
fermentation and food waste as raw materials to eliminate Pb (II) and Cd (II)
from aqueous solution. The results showed that A-HA adsorption of Pb (II)
was superior than Cd adsorption (II). Because of its environmental
friendliness, low cost, and derivation from food wastes as source materials,
the authors concluded that A-HA could be a promising and effective adsorbent
for metal removal (Chen et al., 2022). Researchers discovered that acid
hydrothermal circumstances favor biomass waste breakdown and creation of
AHA precursors such as saccharides, furans, organic acids, and phenols,
whereas alkaline hydrothermal conditions favor AHAs production from these
precursors. The researchers hypothesized and demonstrated enhanced
humification from biomass waste using a mixed acid-alkaline hydrothermal
method in their study (Shao et al., 2022). The conversion of low rank coal into
value-added products such as humic acids (HAs), which are widely used as
fertilisers, is a promising technology. The use of Pakistani Thar lignite for
humic acid extraction has the potential to boost Pakistan's agriculture
business. Fifty-five fungal strains were isolated from Thar lignite and tested
for lignite solubilization efficiency in this study. The isolates M13 and MI had
the maximum ability for lignite solubilization, with a yield of 67.44.3 percent
(w/w). Bio-extracted HA (bHA) had higher nitrogen and oxygen content, but
lower carbon and sulphur content, as compared to chemically extracted HA
(cHA). UV/Vi’s spectroscopy revealed that bHA has a higher E4/E6 value than
cHA, indicating a lower molecular mass and aromaticity, as well as improved
bioactivity (Ghani et al., 2021). Scientists are also looking at extracting humic
acid from a variety of other sources. Subbituminous coal was used as the
starting material for extracting humic acid (HA), which was then used as a
ceramic dispersion. Even if this technology isn't or can't be used in agriculture,
it offers up new possibilities for investigating different sources from which to
extract or produce humic acid (de Souza & Bragança, 2018). The sequential
resin-alkali extraction approach was recently devised to extract humic
compounds from soils, allowing for milder conditions than the traditional
isolation procedure (Timofeevna Shirshova et al., 2006). A one-step approach
was used to create a novel functional colloid-like magnetic biochar (Col-L-
MBC) with excellent dispersibility, using pre-prepared porous biochar as the
skeleton. Notably, AHA obtained from waste biomass using hydrothermal
humification (HTH) technology was found to be rich in functional groups (e.g.,
phenolic-OH, -COOH, etc.), which is conducive to the uniform dispersion of
magnetic nanoparticles on the porous biochar skeleton, resulting in a large
number of active sites for heavy metal ion removal in the soil (F. Yang &
Antonietti, 2020). All of these studies suggest that humic acid formulation has
progressed beyond simply producing humic acids to integrating humic acid
with modern-day technologies to develop novel answers to agricultural
challenges.
Applications of Humic Acids:
Researchers have found that zymo (a dilute mixture of humic and fulvic acids)
when sprayed for eight weeks stimulated higher photosynthetic pigment
accumulation and greater photosynthetic efficiency, from the 5th application
at the beginning of fruit harvest. It was also found to increase the quality of
fruits, reducing number of misshapen and rotten fruits, and also increased
sugar content in fruits was noticed. (Neri et al., 2002) Another research
involved the application of humic acid on the soil a month before plantation.
This improved the nitrogen intake by the corn plant while foliar applications
improved uptake of P, K, Mg, Na, Cu and Zn. It was also found that humic acid
improves the root length of the plants, also increasing fresh and dry weight of
plants.(Khaled & Fawy, 2011) A group of scientists inferred that the foliar
application of humic acid caused a transitional production of dry mass of
durum wheat, this effect was clearly evident for grain yield, spike fertility and
grain protein content during the two years of the study.(Delfine et al., 2005) A
special study on foliar application of humic acid on plants was conducted and
it revealed that bio stimulant effects of humic substances are characterized by
both structural and physiological changes in roots and shoots related to
nutrient uptake, assimilation and distribution (nutrient use efficiency traits). In
addition, they can induce shifts in plant primary and secondary metabolism
related to abiotic stress tolerance which collectively modulate plant growth as
well as promoting fitness. (Canellas et al., 2015) Another group of scientists
studied the foliar application of humic acid on the Canola plant. The results
showed that foliar application of humic acid significantly affected plant height
and highest this parameter was achieved under 2% foliar application of humic
acid and the lowest plant height was obtained under control conditions. Also,
means comparison showed that plant height under 0.5% foliar application of
humic acid and 1% foliar application of humic acid were in a similar statistical
group. The results showed that foliar application of humic acid decreased
nitrogen application in soil, that can be the most important for the non-
pollution of soil by nitrogenous fertilizers. By the application of humic
substance to plants, the growing plants are supplied with food, its application
also results in productive and fertile soil, which increase the water holding
capacity of soil. It plays a pivot role in making the plants more resistant
against drought stress, and also stimulates germination. The application of
humic reduces the requirement of other fertilizers. It also increases crop yield,
soil aeration, and drainage can also be improved by humic, the establishment
of desirable environment for the development of microorganisms. Increase in
the protein and mineral contents of most crops is possible by the application
of humic substances. (Sani, 2014) In another study, foliar application of humic
acid on olive trees were studied. A mixture of humic acid and arginine yielded
fruits with the highest protein content and total chlorophyll, and those treated
with humic acid had the highest anthocyanin content. Foliar application of
humic acid on the ‘Zard’ olive cultivar, it yielded the fruits with the highest
phenol content. (Miri Nargesi et al., 2022) A case study was done on tuber
growth and quality of potato. On foliar application of fulvic acid and the soil
incorporation of humic acid, the weight of the extra-large tubers increased
thereby increasing the total productivity and increased mineral content in soil
and tubers were observed. (Suh et al., 2014) A special study was done on the
cucumber plant, on foliar application of humic acid (3 g/l) on cucumber plants
along with Ecormon (0.45 g/ml) led to positive effect on plant growth, fruit set
and improvement in production. It was also found that the chemical content
percentage (K, P, N, Ca and Mg) increased in the leaves of the cucumber
plants. (El-Nemr et al., n.d.) Another study involved the usage of humic acid
along with phosphorous on lettuce plants, where the interaction of humic acid
with phosphorous improved the nitrogen content of lettuce significantly.
(Cimrin et al., 2005) A research study was conducted on tomato plants
revealing that foliar application of Calcium (15 mM) + humic acids (30 ppm)
resulted in the maximum TSS (5.14 °Brix), vitamin C (25.14), nitrate reductase
activity (6.4), yield (25.36 t ha–1), fruit firmness (3.91 kg cm–2), fruit lycopene
content (2.14) and the lowest blossom end rot incidence (5%). In Finally, humic
acid and calcium chloride application can be helpful for yield improvement and
prevent of decreasing yield. (Environment et al., n.d.) Another group of
scientists inferred that application of humic acid and salicylic acid could be an
effective and low cost approach to ensure seedling establishment and plant
growth in fields affected by soil drought in the early season, especially for the
SC 705 corn hybrid in semi-arid regions.(Bijanzadeh et al., 2019) Scientists
also conducted experiments using onions and found that the application of
humic acid (20 kg per hectare) along with 100% recommended dose of
nitrogen, phosphorous and potassium improved the bulb yield of the onion,
organic carbon content of post-harvest soil and also enhanced the cation
exchange capacity of soil by 18%(Sangeetha et al., n.d.) A group of scientists
conducted an experiment by treating maize seeds with humic acid by pre-
sowing seed treatment method and exogenous or foliar application of humic
acid method in the later stages. Overall, both the methods were effective in
improving salinity tolerance of maize plants and also increased the plant
biomass, chlorophyll contents, mineral nutrients and activities of key
antioxidant enzymes. (Kaya et al., 2018) A group of researchers found that the
foliar applied and root applied humic acid shared the capacity to increase the
concentration of indole-3-acetic acid in roots and cytokinin in shoots. Both
application modes increased the root concentrations of jasmonic acid and
jasmonoyl-isoleucine.(De Hita et al., 2020) Another group of scientists inferred
that on the application of humic acid by the method of drip irrigation, the total
yield of watermelon plants increased, along with increase in fruit quality.
(Salman et al., 2005) Another case study was done on Thymus vulgaris.L
(garden thyme), which revealed that on soil application of humic acid,
essential oil content was enhanced by increase of the humic acid level and its
content ranged from 0.8% (control) to 2.0% (75 g m-2). There was a linear
relationship between humic acid levels and thymol as a major compound. The
humic acid application also enhanced the uptake of N, P, K, Mg and Fe in
garden thyme. (Noroozisharaf & Kaviani, 2018) A study on effect of foliar
application of humic acid on perennial ryegrass (Lolium perenne. L) revealed
that humic acid improved the root and shoot development, except for root
fresh weight. While just 100 mg L−1 improved height, visual quality, nitrogen
(N) content, roots length, and surface of roots, all of HA concentrations were
effective on iron content. (Daneshvar et al., 2015) Another group of
researchers inferred that on application of humic acids increases the
qualitative and quantitative of ‘Askari’ grape compared to untreated plants.
The highest cluster weight, chlorophyll index and TSS were obtained in plants
that were treated with 2.5 gl‐1 humic acid in soil application method.
(Mohamadineia et al., n.d.) Another study conducted by researchers on black
cumin (Nigella sativa L.) showed that different levels of humic acid imposed a
significant effect on number of capsules per plant, number of seeds per
capsule, seed weight, seed yield, biological yield and harvest index. Generally,
it seems that application of nano fertilizer (Pharmks ®) and humic acid due to
having nutritional ingredients and different physiological effects improves N.
sativa performance and reduces environmental pollution.(Safaei et al., 2014)
A special case study conducted on pomegranates revealed that on foliar
application of 6% kaolin significantly decreased cracking in 7 day irrigation
studies while 2 ml l-1 humic acid, 6% kaolin, CB (calcium boron), and the
combination of 6% kaolin/CB and 6% kaolin/2 ml l-1 humic acid reduced
cracking under 14 day irrigation. Fruit weights significantly increased with 6%
kaolin application and 7 d irrigation while 5 ml l-1 humic acid both increased
pomegranate acidity and decreased the flavor index.(Ghanbarpour et al.,
2019) Another group of researchers found that on application of humic acids
with irrigation on corn (Zea mays L.) different levels of humic acid imposed a
significant effect on the yield parameters such as grain yield, biomass, leaf
area index, leaf area duration, height, row number and ear length.(Ghorbani et
al., 2010) Another group of scientists inferred that on foliar application of
humic acid to pungent pepper (Capsicum annuum L.), results revealed that
the highest value of height of plant, numbers of leaf plant per plant, leaf area,
numbers of branch plant-1 , numbers of fruit plant-1 and fruit length were
exhibited by foliar application of humic acid + Zn + B. The highest value of
reducing sugar, total sugar and starch content were obtained by application of
humic acids + Zn + B. (Manas et al., 2014) A special study conducted on
pepper seedling growth under saline conditions revealed that applications of
humic acid and calcium nitrate significantly affected pepper seedling growth.
1000 and 2000 mg kg-1 humic acid and 50 mg kg-1 calcium nitrate
applications increased fresh and dry leaf weight, fresh and dry root weight,
stem diameter, root length and shoot length. (Sönmez et al., 2010) Another
group of researchers inferred that on the foliar application of humic acid
alleviated the symptoms of Cd toxicity by increasing the net photosynthetic
rate (Pn) and the content of photosynthetic pigments. It is concluded that
foliar application of humic acid (1.0 g L−1) can alleviate Cd toxicity and
increase Cd stress tolerance in hybrid pennisetum, thereby lowering the
potential risk of soil Cd contamination. (Song et al., 2020)

Effect of Humic Acid on Microorganisms

Researchers have found that the addition of humic acids into the cultivation
medium extended toleration of the bacteria to higher concentrations of
phenolic compounds and increased the growth of this microbial population as
well. In the case of the yeast, humic acids inhibited the growth. Humic acids
easily form an additive layer on the surface of the investigated
microorganisms. The generated humic acid layer can probably serve as a
transport barrier and thus influence the growth of microbial populations.
(Feifičová et al., n.d.) Another group of scientists concluded that on integrated
addition of humic acid, effective microorganisms (EM) and magnesium
showed that the vegetative parameters, chlorophyll levels, total yield of tuber,
tuber count increased tremendously in potato in clayey soil.(Awad et al., 2007)
A special study performed by scientists inferred that humic acids at a
concentration of 0.1 g/l stimulated the growth of the soil and earthworm’s
intestinal bacteria strains (66 strains out of 161) on Capek’s medium with
glucose (1 g/l), probably, acting as a regulator of the cell metabolism. On the
medium with the humic acid, the intestinal bacteria grew faster than the soil
isolates did.(Tikhonov et al., 2010) Another group of scientists revealed that
application of 100 per cent NPK (nitrogen, phosphorous and potassium)
fertiliser with 20 kg humic acid per hectare recorded higher bacterial
population of 35.8 × 10 5 cfu g-1 while 100 per cent NPK and 30 kg humic acid
per hectare recounted maximum of fungi and actinomycetes (14.0 × 10 3 cfu
g-1 and 15.3 × 104 cfu g -1) population. Humic acid at 20 humic acid per
hectare were found to enhance the catalase activity (164.9 mg of KMnO4
oxidised g -1 20 min-1) and alkaline phosphatase activity of 65.3 m mol p-intro
phenol g-1 hr-1. Humic acid application at 30 kg per hectare significantly
influenced the dehydrogenase activity of 115.9 ig of TPF formed g-1 24 hr-1.
(Sellamuthu et al., 2003) Another group of researchers concluded that plants
that were treated with 300 mg kg-1 of humic acids had the heaviest weights of
both fresh and dry shoots; however, doses of 3000 mg kg-1 were highly
phytotoxic and inhibited the growth of laurel. Vesicular-arbuscular mycorrhizal
(VAM) colonization was only slightly affected by the addition of increasing
concentrations of humic substances to the soil, while the hyphal growth of
Glomus mosseae was reduced. (Vallini et al., 1993) A special study conducted
by researchers using electron paramagnetic resonance spectroscopy showed
that standard humic acids (HA-S) and humic acids (HA) exhibit high anti-
oxidative activity involving free radical scavenging and iron sequestration.
Humic acids additionally promote microbial consortium growth by providing
protection from oxidative stress. (Mileti C et al., 2014) Another group of
scientists found that humic acids increased either NH4/+ or NO2 oxidation and
cell growth of nitrifying bacteria in a dose-independent manner. The
stimulating effect of this fraction of soil organic matter on chemo lithotrophic
ammonia and nitrite oxidizers might be attributed to an increase in microbial
membrane permeability favouring a better utilization of nutrients. (Vallini,
Pera, et al., 1997) A special study on peanut plants revealed that humic acids
increased the productivity of peanut plants and also the number of bacteria
decreased while that of fungi increased after humic acid treatment. The
abundance of Firmicutes in bacteria, Basidiomycota, and Mortierellomycota in
fungi all increased, which have been reported as being beneficial to plant
growth. In contrast, the abundance of Ascomycota in fungi was reduced, and
most of the related genera identified are pathogenic to plants. (Li et al., n.d.) A
group of scientists inferred that addition of humic acids stimulated vegetative
growth of chicory. They also caused significative variations in the numbers of
bacterial heterotrophs and autotrophic nitrifiers in the soil by enhancing the
cell membrane’s permeability to nutrients.(Agnolucci et al., 1996) A group of
researchers conducted an experiment on wheat (Triticum aestivum L.), the
results indicated that mycorrhizal root infection percentage significantly
increased by application of humic acid with AM fungus.(Abou-Aly et al., 2009)
Another group of scientists concluded that humic acid in the form of sodium
humate and fulvic acid markedly increased the growth and efficiency of
nitrogen fixation of Azotobacter chroococcum. The effectiveness of these
substances was more pronounced in increasing the number of cells than
affecting the nitrogen fixation.(Bhardwaj & Gaur, 1970) A special case study
conducted on medicinal plants was done and scientists found that fulvic acid,
humic acid and efficient microorganisms in the production of medicinal plants
contributes on the increase of biosynthesis, secondary metabolites production
such as coumarins, alkaloids, phenylpropanoids and essential oils, as well as
the increase of nutrients absorption, growth and development of species.
(Pereira et al., 2019) Another group of researchers inferred that humic acid
obtained from high quality compost (green compost) suggested that humus-
like substances extracted from compost seem to exert higher stimulative
effects on microbial growth and vegetative biomass production than fossil
humates.(Valdrighi et al., 1995) Another case study was conducted by
scientists and the results showed that the addition of 50, 100, and 500 mg/L
humic acids enhanced ferrous iron (Fe(II)) release in a time-dependent and
dose-dependent fashion under anaerobic conditions. These results suggest
that humic acids can further enhance the microbially mediated release of
sedimentary iron under anaerobic conditions. Furthermore, a microbial
community analysis indicated that the predominant class was changed from
Alphaproteobacteria to Deltaproteobacteria, and clearly increased populations
of Geobacter sp., Paludibacter sp., and Methylophaga sp. were found after
adding humic acids along with the increased release of iron and arsenic. Our
findings provide evidence that humic acids can enhance the microbially
mediated release of sedimentary ferrous iron in an arsenic-affected area.
(Chang et al., 2016) Another group of scientists carried out an experiment on
yeast Candida utilis concluded that the raw extracts of humic acid from peat
and their dialysable and non-dialysable fractions, as well as various aromatic
fractions, exerted no biological activity on the metabolism of C. utilis. These
results show that there were no stimulatory or inhibitory substance was
present. (Mcloughlin & Küster, 1972) Another study on effect of humic acids in
anaerobic microbes was conducted by researchers and the results showed
that with the exception of Methanospirillum hungatei, all pure cultures of
hydrogenotrophic methanogens tested were severely affected by addition of
humic acid. Anaerobic sludge was less affected by the addition of humic acid.
However, a clear gap in the reducing equivalent balance was observed,
probably due to humic acid acting as an alternative electron acceptor and
resulting in reduced methane production in the presence of humic acids.
(Khadem et al., n.d.) Another research study conducted by scientists revealed
that both types of humic acids increased either NH4+ or NO2-oxidation and
cell growth of nitrifying bacteria in a dose-independent manner. The
stimulating effect of this fraction of soil organic matter on chemo lithotrophic
ammonia and nitrite oxidizers might be attributed to an increase in microbial
membrane permeability favouring a better utilization of nutrients. (Vallini,
Agnolucci, et al., 1997) Another group of researchers concluded that both
humic and fulvic acids tended to decrease populations of culturable bacteria
and fungi in the cultivation system, indicating a moderately antibiotic activity.
The addition of saprophytic microorganisms able to use humic substances to
the cultivation system further stimulated the development of the mycorrhizal
fungus, a beneficial species. (Gryndler et al., 2016) A special case study
conducted by researchers inferred that humic acid and seaweed extracts
treatments caused a significant increase in all vegetative growth and yield
characteristics of potato CV desiree. The interaction between treatments also
caused a significant effect and the plants which got a humic acid and sprayed
with mixture of Alga 600 and sea force 2 gave the highest values of vegetative
characters as compared with lowest values of control plants. (Agriculture &
2011, n.d.) Another study conducted by researchers revealed that the
numbers of soil bacteria, fungi and actinomycetes in the HA-NPK1 (humic
acid, nitrogen-phosphorous-potassium) treatment were significantly higher
than the NPK treatment. In conclusion, the application of the activated HA-
NPK in sweet potato production could be used effectively to increase soil
quality, facilitate the growth of sweet potato and increase its yield under
similar conditions to this work. (Wang et al., 2022)

Effect of Humic Acids along with Vermicomposting:

Another group of scientists conducted research on effect of humic acid

produced from earthworm (Eisenia foetida) compost and found that these
humic acids enhance the root growth of maize (Zea mays) seedlings in
conjunction with a marked proliferation of sites of lateral root emergence.
They also stimulate the plasma membrane H-ATPase activity, apparently
associated with an ability to promote expression of this enzyme. In addition,
structural analysis reveals the presence of exchangeable auxin groups in the
macrostructure of the earthworm compost humic acid.(Canellas et al., 2002)
A special case study was conducted on humic acids derived from earthworm-
processed organic wastes and found that the incorporation of both types of
vermicompost¬ derived humic acids, into either type of soilless plant growth
media, increased the growth of tomato angel, cucumber plants significantly, in
terms of plant heights, leaf areas, shoot and root dry weights. Plant growth
increased with increasing concentrations of humic acid incorporated into the
medium up to a certain proportion (Arancon et al., 2002) Another case study
was conducted on humic acids derived from cattle, food and paper-waste
vermicompost. The results showed that substitution of humates ranging from
250-1000 mg kg-1 MM360 increased root growth of marigolds and peppers,
and increased root growth and numbers of fruits of strawberries significantly
(P ≤ 0.05). Leaf areas, plant heights and above-ground dry matter weights
increased considerably in plants grown in pots containing humic acids.
(Arancon et al., 2003)

Disadvantages of Humic Acid:

A group of scientists conducted research on heavy metal accumulation by
humic acids and concluded that humic acids reduced readily soluble and
exchangeable forms of heavy metals in the contaminated soil but increased
their plant-available forms. The greatest increase in the accumulation of
heavy metals was 264. 7 % in the shoot of Festuca arundinacea, with the
bioconcentration factor (BCF) increasing from 0. 30 to 1. 10. These results
suggest that HA amendment could enhance plant uptake of heavy metals.
(Park et al., 2013) Another group of scientists conducted research on effects
of humic acids on a photobacterium (Vibrio fischeri) and found that the toxicity
of lead increased, depending on the concentration of humic acids and the
presence of humic acids the toxicity reduction in the case of the binary
mixture of copper and lead was negligible. (Tsiridis et al., n.d.) Another study
inferred those humic acids caused significant Zn and Pb immobilization
(increased proportion of the residual fraction, extractable only with aqua regia)
in the acid soil, while Cu and Fe were slightly mobilized (increased
concentrations extractable with 0.1 M CaCl2 and/or 0.5 M NaOH). However,
humic acid–metal interactions provoked the flocculation of these substances,
as suggested by the association of the humic acids with the sand fraction of
the soil. (Clemente et al., n.d.)

Conclusion:
To summarise, humic acid (HA) is an essential component and integral part of
the organic structure of soil. Humic acid can help to increase plant
 development and nutrient uptake by reducing unfavourable soil
characteristics. Humic acid application dosages are critical for getting the
most out of it. It is preferable to administer humic acid or humic acid in small
amounts over the course of the crop season rather than a large amount or at
one time.
More than a hundred papers were reviewed in this investigation. Humic acids
play a vital function in boosting the development rate of plants, according to all
of these research publications. On foliar application of humic acid, chlorophyll
content, root length, shoot height, mineral content, fruit size, salt tolerance,
and other traits in plants such as corn, potato, and tomato increased. Because
the interaction between humic acid and microbes was advantageous to both
plants and microorganisms, it was unavoidable in the growth and
development of plants.
We may conclude from this review that humic acid improves soil
characteristics, plant development, and agronomic indices. Coal, lignite, soils,
and organic materials are all sources of HA. Plants require humic acids to
increase cell membrane permeability. Nutrients can enter the plant more
easily as the cell membrane becomes more permeable. Humic acids have the
ability to help soils retain more water.
In fact, when compared to traditional sources, humic acid produced from
green sources was found to be more efficient and effective. Humic acid has a
number of drawbacks, one of which is that it helps plants to absorb heavy
metals from the soil, rendering them unfit for human consumption. Humic acid
can be used as a modern and organic fertiliser and is the greatest option for
chemical fertilisers because its benefits outweigh its drawbacks.

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