Desalination and Water Treatment 53 (2015) 2450–2455

[Link] March

doi: 10.1080/19443994.2013.871582

Pre-treatment of high fat content dairy wastewater using different commercial

lipases

Tejal V. Adulkar, Virendra K. Rathod*

Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India
Tel. +91 22 33612020; Fax: +91 22 33611020; email: [Link]@[Link]
Received 29 July 2013; Accepted 30 October 2013

ABSTRACT

To overcome the shortcomings of direct anaerobic digestion of the dairy waste effluent, an
enzymatic pre-treatment have been employed to significantly overcome the sludge problems
and other drawbacks. This work describes the application of Lipase Z to perform the
enzymatic pre-hydrolysis of a synthetic dairy wastewater containing around 2,000 mg/mL of
fat content. Different process parameters like effect of enzyme loading, temperature, different
concentration of fats and different concentrations of NaCl were optimized for maximum
conversion of fat. The maximum hydrolysis of around 75% is achieved at 0.2% w/v enzyme
loading, 30˚C and 100 mM NaCl concentration for 2,000 mg/mL fats content. It was
contemplated that the enzymatic pre-hydrolysis prior to the anaerobic digestion reduced the
reaction time required for anaerobic degradation, improved biogas formation and
appreciative chemical oxygen demand removal.

Keywords: Dairy wastewater; Anaerobic treatment; Pre-treatment; Enzymes; Lipase

1. Introduction chain of cleaning and washing equipments in the

plant production processing, packaging transportation,
Dairy industry is one of the major industries in
storage and residual milk [2–4]. These dairy wastewa-
India with around 15% growth rate and is estimated
ters are highly concentrated with organic molecules
to cross 150 million tonnes per annum production [1].
like proteins, carbohydrates, lipids and also contain
Lately, the increase in production of milk and milk
large amount of suspended solids, BOD, chemical oxy-
products has also led to immense advancement of
gen demand (COD), oil and grease content. In addi-
dairy industries all over the world [2]. Thereupon, the
tion, it is also composed of detergent and sanitizer.
magnitude of effluent being generated and discharged
All these aspects adding up to the complexity of
from these industries is also increased. Thus, the dairy
wastewater treatment. The organic content in dairy
industry is one of the prime pollution causing indus-
wastewater is a major factor affecting the biodegrad-
tries and pollution potential of this leading economic
ability coefficient of dairy wastewater and induces
sector cannot be neglected. The source of dairy
serious problems like gross land and water pollution,
wastewater is basically from various steps in dairy
bad environmental impact and organic load on
municipal sewage system [3,5]. Oils and grease being
*Corresponding author.

1944-3994/1944-3986 Ó 2013 Balaban Desalination Publications. All rights reserved.

 T.V. Adulkar and V.K. Rathod / Desalination and Water Treatment 53 (2015) 2450–2455 2451

slow degrading compounds cause problems in the Also, there is literature available on the use of enzyme
aeration and pumping system. On the other hand, the for dairy wastewater pre-treatment [5,7,10].
formation of greasy layer on the surface of aeration The objectives of this work are (i) Screening and
tanks interferes with the sedimentation and floccula- utilization of lipase enzymes for dairy wastewater
tion of biomass and hinders the gas transfer required pre-treatment. (ii) To optimize the reaction parameters
for the biological degradation [6]. Also, it exhausts the for maximum FOG conversion. (iii) To analyse the
dissolved oxygen levels of the receiving streams efficiency of the pre-treatment by its reduction in
and moreover generating foul odour creating COD and FOG content.
nuisance conditions. If oil and greases are discharged
without treatment, it can induce severe environmental
pollution by forming oily film on the water surface, 2. Materials and methods
preventing oxygen diffusion and in turn hampering 2.1. Materials
the aquatic life. Thus, it is necessary to generate effec-
tive technique to lessen the negative environmental 2.1.1. Enzymes
impact of dairy wastewater [7,8]. Lipase Z (lipase from Candida rugosa-free form)
The available physical and chemical treatment was obtained as a gift sample from Zytex, Mumbai. It
methods are fast and effective but suffer from various has an activity of 360,000 units per gm.
drawbacks such as requirement of costly reagent,
harsh reaction conditions, hazardous reagents and
higher energy consumption. Therefore, the biological 2.1.2. Wastewater
processes are favoured [3]. In biological methods, the
Dairy wastewater was prepared synthetically by
anaerobic treatment process is preferred over the
diluting different proportions of milk in deionized
aerobic process due to several advantages like
water to get various fat concentrations. The amount of
valuable biogas production, no oxygen required and
fat content was prepared approximately in the range
therefore less energy consumption [1,3]. However,
2,000, 4,000, 6,000 and 8,000 mg/mL higher than the
excessive fats, oils and greases (FOGs) showed inhibi-
normal fat content of dairy wastewater which ranges
tory action on the anaerobic degradation due to
from 200 to 1,200 mg/mL. The milk was bought from a
accumulation of the greasy scum layers limiting the
local dairy, Sharma Dairy, Mumbai. The milk:water
gas transfer and further mixing [6]. Thus, this exces-
ratio of 1:8 was used for the optimization study (2,000
sive FOG leading to process failure was needed to be
mg/mL). This wastewater prepared was used for the
controlled by development of a pre-treatment process
pre-hydrolysis with the help of enzymes and for study-
to accelerate the anaerobic digestion and increasing
ing the characteristics of dairy wastewater. When not in
the degree of stabilization in comparison to classical
use, prepared waste water was stored in deep freezer.
anaerobic digestion without a pre-treatment process
All solvents and reagents are of analytical grade
[1,9].
and purchased from S.D. Fine Chemical, Mumbai,
Amongst all the alternatives, the enzymatic
India.
approach acquired more considerable attention
because of the stringent environmental regulations
due to the clean and eco-friendly enzyme applications
[10]. Eminently, they can be operated under milder 2.2. Analytical methods
conditions with respect to pH and temperature. Thus, 2.2.1. COD
the enzymes are more satisfying due to their biode-
gradability and emerging as a green alternative for It was determined by oxidation of the small amount
other treatment processes [11]. of sample with potassium dichromate and sulphuric
Lipases (E.C.[Link]) fall in to the hydrolases cate- acid (Standard Methods of Water and Wastewater
gory catalysing the lipid/oil hydrolysis to long-chain Examination, APHA, 1995).
fatty acids and glycerol [12]. These enzymes act on the
lipid water interface and particularly hydrolyse the O
2.2.2. Fats, oils and greases
& Gs which usually causes clogging and other severe
problems like fat scum layer at the surface, granular FOGs were determined by the extraction of
mass flotation, etc. in the upflow anaerobic sludge wastewater in a reflux using hexane as a solvent as
blanket reactor [10,13]. Hence, they are of much inter- described in the Standard Methods of Water and
est in high fat content industries like dairy industries. Wastewater Examination, APHA, 1995.
2452 T.V. Adulkar and V.K. Rathod / Desalination and Water Treatment 53 (2015) 2450–2455

The acidity of the raw wastewater was estimated 70

by titrating it with 0.1 N NaOH solution to neutralize
the free fatty acids levels. The characteristics of the 60

dairy wastewater used for the study has been stated

50
in Table 1.

% Hydrolysis
40
2.2.3 Titrimetric analysis
30
The free fatty acids formed in the hydrolysis reac-
tion were evaluated by titrating it with 0.02 N aqueous 20
KOH solution. The free fatty acid formation was used
to supervise the reaction progress. 10

0
2.3. Experimental method Novozym 435 Lipozyme RM Pseudomonas Candida
IM fluorescens rugosa lipase
In a baffled glass reactor (150 mL capacity) of
4.5 cm i.d. equipped with a six-bladed turbine glass
Fig. 1. Screening of different lipases at enzyme loading
impeller, 100 mL of crude wastewater was fed along 0.2% w/v, NaCl concentration 100 mM, temperature 30˚C,
with an enzyme (0.2% w/v) and sodium chloride fat concentration 2,000 mg/mL and agitation speed 200
(100 mM) as an emulsifying aid. The entire reactor rpm.
assembly was immersed in a thermostatic water bath
and maintained at required temperature with an accu-
racy of ± 5˚C. The temperature of the reaction was high molecular FOGs in the dairy wastewater in to
being monitored with the help of a temperature con- fatty acids and glycerol. Four different commercially
troller. The agitation was provided by means of an available lipases namely, Novozym 435, Lipozyme RM
electric motor equipped with speed controlling device. IM (immobilized), Pseudomonas fluorescens and Can-
The reactions were carried out for a maximum period dida rugosa lipase (free) were screened to obtain
of 24 h and at 200 rpm. At regular intervals, a small maximum conversion of triglycerides (fats) in to
sample (2 mL) was withdrawn from the reactor and fatty acid. The pH of the crude wastewater was
transferred to conical flask. Ten millilitres of a 50:50 found to be around 6.5. Fig. 1 shows that amongst
v/v mixture of acetone in ethanol were added to the all Candida rugosa lipase showed satisfying results,
sample to denature the enzyme and freeze the reac- and hence was chosen for the further enzymatic
tion. This mixture was titrated with standard 0.02 M studies.
potassium hydroxide (KOH) solution using
phenolphthalein as an indicator to find the acid value
and percentage conversion. 3.2. Effect of reaction time
The effect of reaction time on hydrolysis of the
3. Results and discusssion synthetic dairy wastewater using lipase Z was inves-
tigated at around 30 ± 2˚C. It can be seen from
3.1. Screening of lipases Fig. 2 that although hydrolytic reaction at first
Lipases from different microbial strains were increased linearly with time, but the rate reduced
screened based on their capability to hydrolyse the considerably as the reaction progressed. The concen-
tration of the free fatty acids formed as the result of
the reaction increased swiftly at the initial 14 h to
Table 1 70.5%, and then very gradually to 74.7% at 24 h and
Characteristics of the dairy wastewater used in this study after that it remained almost constant. It could be
concluded that at first, the interaction between
Parameters Value
enzyme and substrate was ample due to availability
pH 6–6.5 of more enzyme. However, with the further progres-
Density (g/cm3) 1.005 sion, there is possibility that product formed are
Free fatty acids (%) 0.28 blocking the active sites of the enzyme [14,15]. It is
Lipids/FOG (mg/L) 2,000 also predicted that the enzyme seems to be not very
COD (ppm) 42,000
active at acidic pH and all of these resulted in a
Acidity ([Link]/L) 0.4
decreased rate of reaction with time.
 T.V. Adulkar and V.K. Rathod / Desalination and Water Treatment 53 (2015) 2450–2455 2453

80 initial reaction rate was higher at 0.6% w/v of the

enzyme than at 0.2 and 0.4% w/v, the final conversion
70
was found to be lower than these two concentrations.
60 The quick enzyme–substrate complexes formation at
50
higher enzyme loading may led to higher initial reac-
% Hydrolysis

tion and more product formation. However, the for-

40 mation of higher product at start may result in
30
blockage of the active sites at faster rate owing to the
product accumulation leading to decrease in rate as
20 well as conversion. Nevertheless, excess enzyme
10 caused difficulties in assimilation by increased viscos-
ity and thus, hindering the mass transfer [16].
0
0 5 10 15 20 25 30
Time (h)
3.4. Effect of temperature
Fig. 2. Effect of reaction time on hydrolysis at enzyme Sensitivity and reactivity of enzymes are mostly
loading 0.2% w/v, NaCl concentration 100 mM, tempera- dependent on reaction temperature. Thus, the effect of
ture 30˚C, fat concentration 2,000 mg/mL and agitation
speed 200 rpm. temperature on the hydrolysis reaction was studied in
the range from 20 to 60˚C with the optimized 0.2% w/v
enzyme loading as discussed above and 100 mM of
3.3. Effect of enzyme loading
NaCl. The results are depicted in Fig. 4. Generally, the
The enzyme is an important factor economically dissolution rate of substrates and intermediate is
for its commercial application. This effect was studied restricted by lower reaction temperatures; at the same
by varying enzyme loading as 0.1, 0.2, 0.4, 0.6 and time moderate to high reaction temperatures augment
0.8% (w/v) with respect to dairy wastewater used. the reaction rate by decrease in the viscosities of the
The temperature of the reaction was kept at around reaction medium and shortening the reaction time.
30˚C and 100 mM of NaCl was added as an emulsify- Here, reaction temperature of 20–30˚C, showed increase
ing aid. Initially, the conversion of fats to free fatty in the initial reaction rate along with the conversion
acid increased from 0.1 to 0.4% w/v enzyme loading from 60.6 to 74.7%. However, the conversion obtained
i.e. 63.44–77.5% (Fig. 3). On the other hand, further after 24 h for a reaction temperature range of 40–60˚C
increased in loading from 0.6 to 0.8% w/v resulted in decreased from 73.3 to 52.75% (Fig. 4). This can be due
decreased conversion i.e. 70.4–64.85%. Though the to the thermal denaturation of the lipase taking place at

80 80

70 70

60 60
% Hydrolysis

50 50
% Hydrolysis

40 40

30 30

20 20

10 10

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (h) Time (h)
0.1% w/v 0.2 %w/v 0.4% w/v 0.6% w/v 0.8% w/v 20ºC 30ºC 40ºC 50ºC 60ºC

Fig. 3. Effect of enzyme loading on fat hydrolysis at NaCl Fig. 4. Effect of temperature on fat hydrolysis at enzyme
concentration 100 mM, temperature 30˚C, fat concentration loading 0.2% w/v, NaCl concentration 100 mM, fat concen-
2,000 mg/mL and agitation speed 200 rpm. tration 2,000 mg/mL and agitation speed 200 rpm.
2454 T.V. Adulkar and V.K. Rathod / Desalination and Water Treatment 53 (2015) 2450–2455

higher temperature. At higher temperature, the bonds decrease in % hydrolysis with increase in fat acid con-
maintaining the active enzyme conformation are centration. It was found that as the fat content
disrupted and thereby, decreasing the conversion increases from 2,000 to 8,000 mg/mL, the conversion
[16,17]. Therefore, the optimum temperature selected decreased from 74.7 to 56.75%. Though it can be said
was 30˚C, since it was confirmed that at this that the decrease is not very much substantial looking
temperature the enzyme activity was higher and at the very high concentration of the fat and moreover
maximum conversion was obtained. it is noticed that normally the milk processing unit
has fat content of less than 2,000 mg/mL. It can also
be observed that the enzyme do act effectively with
3.5. Effect of fat concentration increased concentration of the FOGs.
The normal fat content in the milk processing unit
was reported to be in the range of 800–1,200 mg/mL
3.6. Effect of NaCl concentration
and depending upon the product manufactured or
process used, the fat content in the dairy wastewater Though lipase showed potential applications in
would vary accordingly. Hence, effect of initial fat degrading oil and fats in wastewater generated by
concentration was studied to determine the activity of dairy industries, emulsifying agents or aids have been
enzyme on different fat content. Fat content in the steadily used to quantify lipase activity, majorly to
range of 2,000–8,000 mg/mL (i.e. 2,000, 4,000, 6,000 amplify the lipid–water interfacial area, as a result of
and 8,000 mg/mL) were evaluated. The enzyme con- which the observed rate of lipase catalysed reaction is
centration and temperature were kept at optimized enhanced [12]. NaCl neutralizes the negative pH gra-
conditions i.e. 0.2% w/v and 30˚C. It can be seen from dient at the interface (created due to attraction of neg-
Fig. 5 that as the concentration of the fats increases, ative charged hydrogen ions by the fatty acids
the conversion is eventually decreased. formed) by reducing the critical micelle concentration
This may be due to the reason that the molecules value and increasing micelles size and thus enhancing
of the enzyme (lipase Z) are coated by the fat mole- the lipase activity [12]. It was seen that regardless the
cules as the fat concentration increases. Thus, render- lipase, runs using NaCl gave higher fatty acid concen-
ing them ineffective towards the hydrolytic reaction trations than runs without the presence of NaCl
and thus decrease in the rate of reaction as well as the (Fig. 6). Further, different concentrations of NaCl rang-
overall conversion. Similarly, the amount of fatty acids ing from 100 to 800 mM were evaluated to determine
formed is constant as the same enzyme loading is the concentration at which it yielded maximum
used for all the fat concentration which resulted in conversion. The reactions were carried out using

80
80
70
70
60
60
50
% Hydrolysis

50
% Hydrolysis

40
40
30
30
20
20
10
10
0
0 0 5 10 15 20 25 30
0 5 10 15 20 25 30 Time (h)
Time (h) 100 mM 200 mM 400 mM 600 mM
8000mg/L 6000mg/L 4000mg/L 2000mg/L 800 mM without NaCl

Fig. 5. Effect of different fats concentration on fat hydroly- Fig. 6. Effect of different NaCl concentrations on fat hydro-
sis at enzyme loading 0.2% w/v, NaCl concentration 100 lysis at enzyme loading 0.2% w/v, temperature 30˚C, fat
mM, temperature 30˚C and agitation speed 200 rpm. concentration 2,000 mg/mL and agitation speed 200 rpm.
 T.V. Adulkar and V.K. Rathod / Desalination and Water Treatment 53 (2015) 2450–2455 2455

0.2% w/v of enzyme and 30˚C. It was seen that the [2] J. Kushwaha, V. Srivastav, I. Mall, An overview of
hydrolysis increased up to 200 mM i.e. from 74.7 to various technologies for the treatment of dairy
76.13% then, it decreased gradually by further NaCl wastewaters, Crit. Rev. Food Sci. Nutr. 51 (2011)
442–452.
concentration from 67.67 to 56.39% (Fig. 6). [3] B. Demiral, O. Yenigun, T. Onay, Anaerobic treatment
Though the initial reaction rate and conversion is of dairy wastewaters: A review, Process Biochem. 40
higher at 200 mM than 100 mM but the difference (2005) 2583–2595.
is not very significant; however, the concentration is [4] W. Janczukowicz, M. Zieliński, M. Debowski,
doubled and hence 100 mM was selected as the opti- Biodegradability evaluation of dairy effluents
originated in selected sections of dairy production,
mized concentration of NaCl. Bioresour. Technol. 99 (2008) 4199–4205.
[5] E. Qamsari, R. Karmanshahi, M. Nosrati, T. Amani,
Enzymatic pre-hydrolysis of high fat content dairy
3.7. Characterization of treated dairy wastewater wastewater as a pretreatment for anaerobic digestion,
Int. J. Environ. Res. 6(2) (2012) 475–480.
The COD of the crude dairy wastewater (before [6] F. Jung, M.C. Cammarota, D.M.G. Freire, Impact of
pre-treatment) was found to be around 42,000 ppm enzymatic pre-hydrolysis on batch activated sludge
which was reduced to 12,000 ppm when the wastewa- systems dealing with oily wastewaters, Biotechnol.
ter was treated at the optimized reaction conditions Lett. 24 (2002) 1797–1802.
[7] M. Leal, D. Freire, M. Cammarota, G. Sant Anna Jr.,
and parameters as discussed above. Thus, it could be Effect of enzymatic hydrolysis on anaerobic treatment
said that after the enzymatic pre-treatment of the syn- of dairy wastewater, Process Biochem. 41 (2006) 1173–
thetic dairy wastewater using lipase Z, 72% of COD 1178.
removal was observed. [8] M.P. Prasad, K. Manjunath, Comparative study on
biodegradation of lipid rich wastewater using lipase
producing bacterial species, Indian J. Biotechnol. 10
4. Conclusion (2011) 121–124.
[9] M.K. Stenstrom, S. Fam, G.S. Silverman, Analytical
The present work evaluates the enzymatic methods for quantitative and qualitative determination
pre-hydrolysis of synthetic dairy wastewater using of hydrocarbons and oil and grease in water and
wastewater, Environ. Technol. Lett. 7 (1986) 625–636.
lipase Z prior to the anaerobic degradation. The fat [10] E. Pereira, A. Mendes, A. Furigo Jr., H. Castro, Anaer-
content of the dairy wastewater was kept at around obic biodegradability of dairy wastewater pretreated
2,000 mg/mL. The various process parameters with porcine pancreas lipase, Braz. Arch. Biol. Tech-
affecting the rate of this enzyme catalysed pre-hydro- nol. 53 (2010) 1279–1284.
[11] W. Adam, M. Lazarus, C. Saha-Mollera, O. Weichold,
lysis reaction were investigated. As justified by the
U. Hoch, D. Haring, P. Schreie, Biotransformation
results, the enzyme loading was selected to be 0.2% with peroxidase, Adv. Biochem. Eng. Biotechnol. 63
w/v and a temperature of 30˚C was selected to be (1999) 73–107.
optimal as above this temperature the yield decreased [12] A. Mendes, H. Costra, Effect on the enzymatic hydro-
subsequently due to instability of the enzyme at lysis of lipids from dairy wastewater by replacing
gum Arabic emulsifier for sodium chloride, Braz.
higher temperature owing to its thermal denaturation.
Arch. Biol. Technol. 48 (2005) 135–142.
NaCl which was used as an emulsifying aid improved [13] L. Masse, K. Kennedy, S. Chou, Testing of alkaline
the enzyme activity and thus the rate of reaction by and enzymatic pre-treatment for fat particles in
around 30% as it provides appropriate reaction condi- slaughterhouses wastewater, Bioresour. Technol. 77
tion required for the interface activation where the (2001) 145–155.
actual lipase reaction takes place. The total amount of [14] M.S. Puthli, V.K. Rathod, A.B. Pandit, Enzymatic
hydrolysis of castor oil: Process intensification studies,
NaCl required was also optimized. Under optimal Biochem. Eng. J. 31(1) (2006) 31–41.
reaction conditions, reaction conversion of 75% was [15] V.K. Rathod, A.B. Pandit, Effect of various additives
observed along with 72% COD removal. on enzymatic hydrolysis of castor oil, Biochem. Eng. J.
47(1–3) (2009) 93–99.
[16] N. Gharat, V.K. Rathod, Enzyme catalyzed transesteri-
References fication of waste cooking oil with dimethyl carbonate,
J. Mol. Catal. B: Enzym. 88 (2013) 36–40.
[1] R.U. Rani, J.R. Banu, S.A. Kumar, S. Kaliappan, I.T. [17] S.M. Radzi, W.A.F. Mustafa, S.S. Othman, H.M. Noor,
Yeom, Low temperature thermo-chemical pretreatment Green synthesis of butyl acetate a pineapple flavour
of dairy waste activated sludge for anaerobic digestion in a lipase catalyzed reaction, W.A.S.E.T. 59 (2011)
process, Bioresour. Technol. 103 (2012) 415–424. 677–680.