Biotechnology and Bioprocess Engineering 25: 132-140 (2020)
DOI 10.1007/s12257-019-0302-4
RESEARCH PAPER
Enhancement of Hydrolysis and Biogas Production of Primary
Sludge by Use of Mixtures of Protease and Lipase
Jovale Vincent Tongco, Sangmin Kim, Baek-Rock Oh, Sun-Yeon Heo, Joonyeob Lee, and Seokhwan Hwang
Received: 24 July 2019 / Revised: 9 September 2019 / Accepted: 21 September 2019
© The Korean Society for Biotechnology and Bioengineering and Springer 2020
Abstract This study aims to improve the hydrolysis and
degradation of primary sludge by using wild-type enzymes
(protease and lipase) and establishing the optimal enzymatic
cocktail ratio. Primary sludge from three wastewater
treatment plants (WWTPs) in Korea (Ulsan, Pohang, and
Busan) were subjected to enzymatic hydrolysis. Protease
and lipase were isolated from enzyme-producing micro-
organisms cultured from secondary sludge collected at 8
different digester sites in Korea. Primary sludge degradation
through enzymatic hydrolysis was monitored by measuring
the reduction in the volatile suspended solids (VSS)
content of the sludge and enzyme cocktail mix for 72 h at
40oC and pH 7.0. The enzymatic cocktail of Ulsan primary
sludge treated with protease to lipase at a ratio of 1:3 was
found to be optimal at 33.3% VSS reduction. Biochemical
methane potential (BMP) tests were employed to the optimal
enzyme cocktail to measure the potential of the hydrolyzed
substrate for further degradation (VSS reduction) and
bioconversion to biogas using 125 mL serum bottles as
anaerobic reactors for 30 days. BMP tests showed that
there was an increase in biogas production by 84.1%,
methane production by 89.8%, and methane yield by 9.6%.
Methane production rate was also increased. The significant
VSS concentration reduction and higher biogas and
methane yield of the enzyme-treated primary sludge
correlate to the fact that the complex polymeric organic
materials were degraded leading to efficient utilization by
the microorganisms in the anaerobic digestion process.
Jovale Vincent Tongco, Sangmin Kim, Joonyeob Lee, Seokhwan Hwang*
Division of Environmental Science and Engineering, Pohang University of
Science and Technology (POSTECH), Pohang 37673, Korea
Tel: +82-54-279-2282; Fax: +82-54-279-8299
E-mail: shwang@postech.ac.kr
Baek-Rock Oh, Sun-Yeon Heo
Microbial Biotechnology Research Center, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), Jeong-eup 56212, Korea
pISSN 1226-8372
eISSN 1976-3816
Keywords: primary sludge, enzyme cocktail, hydrolysis,
anaerobic digestion, protease, lipase
1. Introduction
Sludge is the semi-solid residual material of wastewater
treatment and is classified into two main types; namely
primary and secondary sludge. Primary sludge is the
resulting material when suspended solids and organics are
captured through gravitational sedimentation in a primary
treatment process [1]. Secondary treatment processes
including activated sludge process utilize microorganisms
for the consumption of the complex organics in the
wastewater. The microorganisms feed on the readily
available organic materials in the tank and then flow into a
secondary clarifier where it settles down and removed as
secondary sludge [2].
The two types of sludge differ in terms of biogas
production potential when subjected to anaerobic digestion.
Primary sludge contains higher biogas production potential
because it is just physically separated and captured by
gravity. Thus, the bulk of its energy content has not yet been
consumed. On the other hand, secondary sludge possess a
lower biogas potential because the microorganisms involved
in the secondary treatment process already used up and
consumed the majority of the energy content of the sludge.
Different types of enzymes are effective biocatalysts in
hydrolyzing sludge. Wild-type enzymes refer to enzymes
from microorganisms with particular phenotypes encoded
by alleles commonly found in natural populations in the
environment. The enzymes commonly used for pretreating
sludges are lysozyme [3,4], proteases, amylases, cellulases,
and lipases [5,6].
The biological pretreatment of sludge is more advan-
tageous compared to either chemical or physical pretreat-
Enhancing Hydrolysis of Sludge by Enzyme Mixture
ments because it minimizes unwanted side products which
lead to pollution and does not require specialized or
sophisticated equipment to implement [7]. It can be
classified into 2 categories: (1) introduction of endogenous
or commercial enzymes prior to anaerobic digestion [6];
(2) and incorporation of specific bacteria that produce the
target enzymes [8,9]. Pretreatment using this method
improved sludge degradation and increased biogas and
methane production [10].
Commercial enzymatic preparation is likely to be impro-
bable because it is expensive and incurs high maintenance
costs [10]. Thus, the improvement of the endogenous
enzymes or culturing extraneous enzyme-producing micro-
organisms is more realistic and beneficial. Thermophilic
microorganisms had been widely used for sludge hydrolysis
and subsequent thermophilic anaerobic digestion [8]. A
mixture of protease and amylase (i.e., ratio of 1 to 3), for
example, increased biosolids hydrolysis efficiency from
10% (control test) to 68.43% at the temperature of 50°C
[6]. However, the majority of anaerobic digestion processes
are mesophilic and pretreatment by mesophilic microbial
enzymes has not been thoroughly researched [11,12].
Furthermore, carbohydrates, one of the major components
of primary sludge, are very susceptible to degradation at
mesophilic conditions, compared to proteins and lipids
[13,14]. It would be, therefore, a novel approach if enzymes
can be obtained from natural microorganisms living in
various engineered systems treating such high crude
organics, prepared as a mixed form, and used for pretreating
the target organics easily. The uses of enzymes are known
to have an economic advantage against the energy require-
ments in typical physicochemical pretreatment techniques
(e.g., ozone, acid or alkali, ultrasonication, microwave,
and thermal treatments). These high-energy pretreatment
processes frequently require high capital cost, incur toxic
side products, and more costs and challenges on scaling up
[15].
The efficiency of the enzymatic hydrolysis of primary
sludge can be attributed to the degradation rate and product
quality (substrates broken down into simpler constituents
compared to other treatment options). The lifetime of the
enzymes also contributes to the overall efficiency of the
hydrolysis process because it dictates the amount of
accumulated disintegrated products in which biogas
production is dependent. Employing an enzyme with high
enzymatic efficiency with a short lifetime can produce the
same effect as utilizing an enzyme with low enzymatic
efficiency with long lifetime. The ideal scenario would be
the optimization and screening of enzymes that exhibits
very high enhancement rate and long lifetime [16]. Using
primary sludge does not pose any serious problems in terms
of enzymatic process efficiency as compared to secondary
133
(waste activated) sludge and even lignocellulosic rich
substrates. The reason is that the nature of primary sludge
is simpler in terms of the structure and availability of the
organics. The organic compounds (carbohydrates, proteins,
and lipids) in the substrate are easily accessible (ease of
contact) with the active sites of the enzymes, thus improving
the activity of the enzyme in hydrolyzing the substrate.
This leads to a shorter contact time requirement in using
primary sludge as a substrate which could be in just a
matter of hours as compared to days with secondary sludge
or even several weeks to several months in the case of
lignocellulosic biomass [17].
Therefore, the main objective of this research was to
investigate the effect of using different mixtures of protease
and lipase on the hydrolysis of primary sludge as well as
biomethane potential (BMP). Strains isolated from 8
different wastewater treatment sites across Korea were
cultivated to produce protease and lipase. The effect of the
enzyme mixture (i.e., cocktail) consisting of protease and
lipase on the hydrolysis and degradation of primary sludge
was investigated. After hydrolysis, the potential of
subsequent mesophilic anaerobic digestion through BMP
tests was observed.
2. Materials and Methods
2.1. Preparation of protease and lipase enzymes
Protease (P) and lipase (L) were obtained from the Korea
Research Institute of Bioscience and Biotechnology
(KRIBB). Wild-type enzymes were isolated from enzyme-
producing microorganisms cultured from secondary sludge
collected at 8 different Korean wastewater treatment plants
located in Seoul, Busan, Masan, and Jecheon, as shown in
Table 1. To isolate bacteria producing protease and lipase,
1 g secondary sludge sample was serially diluted in sterile
PBS buffer (pH 6.8). Then, 100 µL samples were spread
onto the surface of nutrient broth agar (Difco, Fischer
Scientific, NH, USA) containing 10 g/L skin milk and 5 g/L
olive oil (Sigma, MO, USA) with 1 mg/L rhodamine B
(Sigma, MO, USA). Samples for protease were cultured at
25°C for 2 days to form a clear zone for protease producing
bacteria screening. Additionally, samples for lipase were
cultured at 25°C until the strains with lipolytic activity
showed shiny orange halos under ultraviolet light (350 nm).
Single colony isolation of each strain was obtained by
performing subcultures 3 times and each of the isolated
single colony was incubated at 25°C for 48 h in nutrient
broth and stored in a 20% glycerol stock solution at –80°C.
Genomic DNA was obtained by using a Genomic DNA
Purification Kit (Invitrogen, USA). The 16S rRNA gene
was amplified by PCR using two universal primers, 27F
134
Table 1. Configurations of the wastewater treatment plants for microbial sampling
Parameters Nanji Seonam Tancheon
Location (City) Seoul Seoul Seoul
Daily flow rate (m3/day) 1,000,000 2,000,000 900,000
Treatment process* MLE MLE MLE
Operational mode** CSTR CSTR CSTR
Operating Temperature (°C) 25 25 24
*MLE = Modified Ludzack-Ettinger, CAS = Conventional Activated Sludge, NPR = Nitrogen and Phosphorus Removal
**CSTR = Continuous Stirred-Tank Reactor, SBR = Sequencing Batch Reactor
(5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-
GGTTACCTTGTTACGACTT-3′). After PCR, the sequence
was compared with valid 16S rRNA gene sequences from
GenBank (www.ncbi.nlm.nih.gov/blast) [18]. Phylogenetic
analysis was performed using MEGA6 [19] with the
neighbor-joining method [20]. The resulting bootstrap
values were calculated based on 1000 replicates [21].
Each seed cells for protease and lipase production were
prepared in 1 L baffled flasks containing 300 mL of
nutrient broth. Flasks were incubated at 30°C for 24 h, and
cultures (10% v/v) were subsequently inoculated into the
fermentor. The protease producing fermentation process
was conducted in a 5 L stirred-vessel system (Kobiotech,
Incheon, Korea) containing 3 L protease production medium
(20 g/L Fructose, 0.1 g/L KH2PO4, 0.1 g/L K2HPO4, 5 g/L
yeast extract, and 1 g/L CaCl2) at 30°C, agitation speed
300 rpm, and aeration rate 1 vvm. The lipase producing
fermentation process was conducted in a 5 L stirred-vessel
system (Kobiotech, Korea) containing 3 L lipase production
medium (10 g/L oleic acid, 0.3 g/L KH2PO4, 0.3 g/L
K2HPO4, 0.5 g/L (NH4)2SO4, 1 g/L yeast extract, 0.4 g/L
MgSO4, 0.7 g/L MgCl, and 0.5 g/L CaCl2) at 30°C,
agitation speed 600 rpm, and aeration rate 1 vvm. The pH
was maintained at pH 7.0 ± 0.2 using 2 M NaOH or HCl.
Cell growth was monitored by measurement of OD600nm
using a UV-Vis spectrophotometer (Ultrospec 3100 Pro;
Amersham Biosciences, IL, USA). Culture broth was used
for enzyme activity and metabolites analysis. Three
independent experiments were performed and then averaged.
2.2. Enzyme activity determination
Protease activity was determined by incubating 0.1 mL of
the cell-free supernatant with 1 mL of 0.6% (w/v)
Hammarsten casein (Sigma) in 50 mM sodium phosphate
(pH 7.0) at 50°C for 30 min by a modified method of Folin
and Ciocalteu [22]. The reaction was stopped by adding
1 mL of 0.44 M trichloroacetic acid (TCA), followed by
centrifugation at 13,000 rpm for 10 min. After the addition
of 0.625 mL of 0.55 M sodium carbonate solution to
0.25 mL of the supernatant, 0.125 mL of Folin reagent was
added to the mixture and left for 30 min, after that the
tyrosine liberated was measured at 660 nm. One unit of
Biotechnology and Bioprocess Engineering 25: 132-140 (2020)
Jungnang Gangbyeon Suyeong Jecheon Masan
Seoul Busan Busan Jecheon Masan
1,710,000 450,000 452,000 70,000 500,000
CAS MLE CAS NPR NPR
SBR CSTR SBR SBR SBR
25 23 23 23 22
protease activity was defined as the amount of enzyme that
released 1 μg tyrosine per min.
Lipase activity was determined with p-nitrophenyl
palmitate (pNPP) as substrate [23]. The reaction mixture
consisted of 100 µL of 8 mM substrate in 2-propanol,
20 µL of lipase solution, and 880 µL of reaction buffer
containing 50 mM Tris-HCl (pH 8.0), 0.1% gum Arabic
and 0.2% deoxycholate. After 20 min of incubation at
37°C, the reaction was stopped by the addition of 1 mL of
2 M NaOH, and the p-nitrophenol released was monitored
spectrophotometrically at 405 nm, using a standard curve.
One unit of lipase activity was defined as the amount of
enzyme that released 1 μmol p-nitrophenol per min.
2.3. Primary sludge sampling and enzyme treatment
Primary sludge samples were collected and obtained from
three WWTPs in Korea (Ulsan, Pohang, and Busan). After
collection, the samples were sealed up in 200 mL sterile
plastic bottles, transported to the laboratory as soon as
possible, and stored at 4oC. The sludge samples were
filtered by 0.60 mm sieve to achieve uniformity in particle
size and remove unwanted huge particles such as sand and
gravel. The total and volatile solids (TS and VS) along
with the total and volatile suspended solids (TSS and
VSS), carbohydrate, protein, and lipid content of the sludge
samples were measured prior to storage.
The solids concentration of primary sludge was analyzed
according to Standard Methods [24]. Carbohydrates
concentration was measured using the phenol-sulfuric acid
method [25]. The protein and lipid concentrations were
measured by using the Kjeldahl method [24] and gravimetric
method using chloroform: methanol extraction (1:2 v/v)
[26], respectively. All sludge analysis measurements were
carried out in duplicates.
The enzyme cocktail was prepared by mixing protease
and lipase into different ratios (protease only (P), protease :
lipase = 3:1 (P:L = 3:1), P:L = 1:1, P:L = 1:3, and lipase
only (L); w/w, dry weight basis). The enzymes were
normalized to a concentration of 1 g VS/L and the sludge
at 1 g VSS/L and subsequently mixed. Phosphate buffer at
pH 7.0 was added to the mixture to prevent sudden
changes in pH that may limit the activity of the enzymes.
Enhancing Hydrolysis of Sludge by Enzyme Mixture
The resulting mixtures were placed in a 50 mL conical tube
and incubated at 40oC for 72 h with constant shaking. The
setup was run in duplicates.
The hydrolysis of primary sludge was monitored every
24 h. The VSS concentration of the enzyme cocktail and
sludge mixture was measured each day using Standard
methods [24] and ultimately used to calculate for the VSS
reduction of the enzyme and sludge mixture. The
measurement of VSS reduction has been the standard
method in monitoring the degradation of sludge for years
[6,27-29]. The optimal enzyme cocktail ratio was obtained
after 72 h and then inactivated by UV light for 30 min [30]
prior to biochemical methane potential (BMP) tests.
2.4. Biochemical methane potential (BMP) test
BMP tests were carried out to evaluate the enzymatic
treatment effect of the optimal wild-type enzyme ratio
selected in the former hydrolysis assay. The tests were
carried out in 125 mL serum bottles with a working
volume of 70 mL. All bottles were seeded with digestate
obtained from a 3.5 L continuous lab-scale mesophilic
anaerobic digester treating sewage sludge. The substrate to
inoculum ratio was 1 g VSs/g VSSi, and each bottle
contained 1.0 g VS of substrate. Basal medium was
prepared and added to each serum bottle according to
literature [31]. After the addition of the substrate to the
inoculum and medium, mixed CO2/N2 gas was used to flush
out residual air in the headspace, ensuring an anaerobic
environment. Afterward, the bottles were incubated at
37oC and pH 7.5 for 30 days. Biogas production was
measured periodically by using an electronic digital
manometer (Leo 2, Keller, Switzerland) and the pressure
readings were converted to volume using the ideal gas
equation [32]. Gas samples from the headspace were
collected and analyzed on the last day for the determination
of biogas composition by using a gas chromatograph (6890
Plus, Agilent, Palo Alto, CA, USA) equipped with HP-5
capillary column and a thermal conductivity detector. The
same set of treatments were also applied to the control
(sludge only) and inoculum blank (without substrates). The
BMP tests were also carried out in duplicates, corresponding
to the hydrolysis assay.
3. Results and Discussion
3.1. Isolation and preparation of enzymes
Secondary sludge samples were collected at 8 different
digester sites in Korea, and the protease and lipase
producing bacteria were isolated and identified based on
NCBI blast results of the 16S rRNA gene sequence. The
NCBI blast program showed that the sequence of protease
135
(PA21) and lipase (LA96) producing bacteria had high
similarity (> 99%) to that of Bacillus amyloliquefaciens
DSM7T and Burkholderia vietnamiensis LMG 10929T,
respectively. In the 16S rRNA phylogenetic tree (Fig. 1),
B. amyloliquefaciens PA21 and B. vietnamiensis LA96 were
located in a well-supported monophyletic group including
the Bacillus and Burkholderia type strain, respectively.
B. amyloliquefaciens PA21 and B. vietnamiensis LA96
were fermented in 5 L jar fermentors. Fig. 2 shows the
enzymatic activity of the resulting protease and lipase and
the cell growth of B. amyloliquefaciens PA21. For the
fermentation of protease producing B. amyloliquefaciens
PA21, the maximum production of protease was optimal at
325.3 U/mL at 18 h and cell growth was OD 5.2 at 24 h. While
for the fermentation of lipase producing B. vietnamiensis
LA96, the measurement of cell growth was unachievable
due to the turbidity of the fatty acids in the culture broth.
Lipase activity was optimal at 21.9 U/mL at 18 h.
Table 1 shows the configurations and parameters of the
8 different digester sites treating secondary sludge as sources
of enzyme-producing microorganisms. The operating
temperatures are well within the range of 22 - 25oC. This
means that the temperature, origin, and type of wastewater
have no specific effects on the composition of protease and
lipase during production, especially if the enzymes were
prepared from microorganisms coming from the same
genus (i.e. Bacilli and Burkholderia for protease and lipase,
respectively). The types of protease and lipase produced
from the same genus have the same range of optimal
temperatures and pH in which they have maximum activity
and stability [33,34]. The preparation and storage of the
enzymes was done at a low temperature (maintained at
25oC for preparation and -80oC for storage) to prevent
protein denaturation and preserve biological properties
[34]. The obtained protease and lipase were used for
enhancing primary sludge hydrolysis.
3.2. Characterization of primary sludge
Primary sludge sourced from the three WWTPs were
analyzed and the physicochemical characteristics and
composition were measured, as shown in Table 2. The
primary sludge was semi-solid during characterization and
needs to be filtered and homogenized prior to hydrolysis.
Ratios of total suspended solids (TSS) and volatile solids
(VS) to total solids (TS) were 83.6% and 76.7%, respectively
for the Ulsan primary sludge, 96.3% and 69.8%, respectively
for the Pohang primary sludge, and 81.9% and 77.6%,
accordingly for the Busan primary sludge. A high ratio
between VSS to TSS indicated that 85.7%, 72.0%, and
89.5% of the suspended materials in the primary sludges
from Ulsan, Pohang, and Busan were respectively due to
the presence of organic materials. The measurement of
136 Biotechnology and Bioprocess Engineering 25: 132-140 (2020)

Fig. 1. Neighbor-joining phylogenetic tree of Bacillus amyloliquefaciens PA21 (A) and Burkholderia vietnamiensis LA96 (B) based on
16S rRNA gene sequence data. The numbers at each internal branch show the bootstrap values (%) for the nodes calculated by 1000
replicates.

degradation of the solids content over time (in the case of
this study, VSS reduction) is a key parameter in every sludge
degradation studies [6,35]. Primary sludge is composed
mainly of carbohydrates, proteins, and lipids. The VS
content of any substrates in anaerobic digestion is the basis
of the amount of organic compounds present. Using
standard methods to quantify the amount of carbohydrates,
proteins, and lipids in terms of VS gives a good estimate of
the amounts of each component in sludge [27]. From
Table 2, it can be seen that protein is the most predominant
Enhancing Hydrolysis of Sludge by Enzyme Mixture 137

VS, also known as refractory volatile solids, are comprised

of recalcitrant organics such as lignin, pesticides, PAHs,
certain phenols i.e. Benzene and Toluene, and heterocyclic
compounds containing nitrogen which has been included
into the wastewater treatment process [37].

3.3. Effect of different enzymatic ratios on hydrolysis of

primary sludge
The enzymatic treatment of primary sludge was employed
for the analysis of the degree of degradation of the organics
present in terms of VSS reduction. In the case of selecting
the optimal enzyme cocktail, the primary sludge samples
Fig. 2. Production of protease and lipase during fermentation by from the three WWTPs were treated with different enzyme
Bacillus amyloliquefaciens PA21 and Burkholderia vietnamiensis ratios. Fig. 3 shows the time profile of the VSS measurements
LA96. Gray bar, protease activity; White bar, lipase activity; for the optimal enzyme cocktail mix (Ulsan primary sludge
Closed circles, cell growth (PA21); Cell growth of LA96 was
unachievable. treated with different enzyme ratios) set at 72 h, clearly
presenting the reduction in VSS as time goes by. The
volatile suspended solids serve as the measure of the
Table 2. Total and volatile solids, carbohydrates, proteins, and amount of organics in primary sludge (excluding soluble
lipids content of primary sludge taken from Ulsan, Pohang, and
Busan wastewater treatment plants
organics i.e. the water-soluble enzymes). As the enzymes
act upon the organic compounds, the VSS will be reduced
Parameters (g/L)* Ulsan Pohang Busan
because the polymeric proteins and lipids in the primary
TS 15.9 ± 2.1 18.9 ± 0.7 11.6 ± 0.2
sludge will be disintegrated into their corresponding simpler
VS 12.2 ± 0.4 13.2 ± 0.1 9.0 ± 0.0
TSS 13.3 ± 1.8 18.2 ± 0.2 9.5 ± 0.1
monomeric units, thus making them soluble. In turn, they
VSS 11.4 ± 0.1 13.1 ± 0.0 8.5 ± 0.0
will be included in the soluble organics that pass through
Carbohydrates 3.5 ± 0.2 4.4 ± 0.1 2.7 ± 0.1 the filter leaving the non-disintegrated organics behind and
Proteins 6.6 ± 0.8 6.5 ± 0.1 4.4 ± 0.0 registers as VSS after drying at 105oC and ignition at
Lipids 0.7 ± 0.1 1.6 ± 0.0 0.9 ± 0.0 550oC [24]. The highest VSS reduction was notable on the
*Expressed in Mean ± S.D. (standard deviation)
P:L = 1:3 ratio hydrolyzed sludge followed by lipase only
treated primary sludge, corresponding to 33.3% and 30.4%
VSS reduction, respectively. The other treatment ratios,
component at 6.6 ± 0.8 g/L (54.4% of VS), followed by
carbohydrates at 3.5 ± 0.2 g/L (28.7% of VS), then lastly
lipids at 0.7 ± 0.1 g/L (5.3% of VS) for the Ulsan primary
sludge. The primary sludge from Pohang WWTP afforded the
following carbohydrates, proteins, and lipids concentration;
4.4 g/L (33.6% of VS), 6.5 g/L (48.9% of VS), and 1.6 g/L
(12.1% of VS), respectively. On the other hand, primary
sludge from Busan WWTP showed carbohydrates, proteins,
and lipids concentration; 2.7 g/L (30.1% of VS), 4.4 g/L
(48.9% of VS), and 0.9 g/L (10.0% of VS), respectively.
This reflects the results of other studies in which the
primary sludge they utilized also consists of proteins as the
most abundant organic component followed by carbohydrates
and then lastly lipids [27,35,36]. The combined amounts of
carbohydrates, proteins, and lipids in the primary sludge,
also known as the biodegradable volatile solids, was found
to be 10.8 g/L for the Ulsan primary sludge, 12.5 g/L for
the Pohang primary sludge, and 8.0 g/L for the Busan Fig. 3. Time profile of volatile suspended solids of the optimal
wild-type enzyme cocktail and Ulsan primary sludge mix set for
primary sludge. This portion comprises the 88.4%, 94%, 72 h. Protease : Lipase ratio of 1:3 (P:L = 1:3) was optimal after
and 88.9% of the VS of Ulsan, Pohang, and Busan primary 72 h of hydrolysis at the volatile suspended solids (VSS)
sludge, respectively. The remaining organics present in the reduction of 33.3%.
138
including the protease only treated primary sludge can be
seen aggregating at almost similar VSS concentration after
72 h, at around 1.8 g/L (27 – 30% VSS reduction). The
VSS reduction can be attributed mainly to substrate
degradation only because the pH (~7.0) and temperature
(40oC) used in the study for both protease and lipase (pH
7.0 – 9.0, 40-60oC) are well within high in terms of their
enzymatic activity and thermal and chemical stability
[38,39]. There is no reported interaction between protease
and lipase in literature. A study on the concomitant
production of protease and lipase for industrial use proved
that it is possible to purify and store the enzymes together
in a single mixture [40].
Both the Pohang and Busan primary sludge also showed
1:3 ratio of protease to lipase to have the maximum VSS
reduction at 28.2% and 26.6%, respectively. Only the
Ulsan primary sludge was subjected to BMP tests because
it yielded the highest VSS reduction at 33.3%, as stated
previously. This shows that primary sludge samples from
different sources with similar organic compositions undergo
the same reactivity with enzymes to the extent that they all
shown to have the optimal enzyme cocktail ratio of 1:3
(Protease : Lipase) producing the maximum VSS reduction.
The amount of proteins exceeds the amount of lipids in
the primary sludge sample greatly but the optimal enzyme
cocktail ratio of P:L = 1:3, which contains higher amount
of lipase, was the most efficient in breaking down the
sludge in terms if VSS. This can be explained by the nature
of the degradation of lipids in the presence of lipase. The
hydrolysis of lipids leads to the production of volatile and
long-chain fatty acids in the presence of lipase. However,
lipid hydrolysis using lipase is inhibited by the accumulation
of these products. The interactions between the enzyme
and the substrate are interfacial, meaning the lipase needs
a target interface to be activated. The reaction mechanism
of lipid hydrolysis is therefore reliant on the quality and the
concentration of the interface [41]. As the fatty acids
increase due to lipid degradation, a higher concentration of
lipase compared to protease might be required to fully
hydrolyze the lipids present in the primary sludge, thus
leading to the optimal enzyme cocktail ratio. Lipase and
protease only treatments were shown to be less efficient in
degrading the organics in primary sludge because they only
hydrolyze their intended organic targets. The enzyme
cocktail was inactivated by UV light prior to BMP tests to
completely reduce and halt the enzymatic activity of the
protease and lipase [30]. Inactivation was deemed necessary
to prevent unwanted reactions and effects in the BMP tests.
3.4. Biochemical methane potential (BMP) tests
The wild-type enzyme cocktail ratio (P:L = 1:3) hydrolyzed
sludge was further subjected to BMP tests for subsequent
Biotechnology and Bioprocess Engineering 25: 132-140 (2020)
energy conversion and to measure the effect of degradation
of the protein and lipid components of primary sludge on
biogas and methane production and content. The cumulative
biogas production was calculated by subtracting the
inoculum blank reading from the enzyme hydrolyzed
primary sludge readings. The biogas and methane (CH4)
production values obtained were 20.8 ± 1.1 mL and 11.8 ±
0.6 mL, respectively. Methane content for the duplicate
trials was 56.8 ± 0.1% and 51.85 ± 0.0% for the inoculum
blanks. The BMP test showed an increase of 84.1% biogas
production and an increase of 89.8% in methane production
(Fig. 4) after 30 days of incubation time between the
untreated and treated primary sludge. As can be observed,
the effect of hydrolyzing primary sludge using enzymes
not only increased biogas production but also methane
yield at 9.6%. The conditions of the BMP tests were set at
pH 7.5 and 37oC to make certain that the microorganisms
are in their optimum environment and also to ensure that
there is no further unwanted degradation occurring (i.e.
thermal degradation and/or degradation due to pH change)
which could ultimately affect BMP readings. Non-linear
curve fitting using the Growth/Sigmodal Hill function [42]
of the statistical software OriginPro 8.0 (Origin Lab, USA)
was done to calculate the rate of methane production (k)
from day 0 to 10. This part of the test showed a steady
increase in biogas production for both the treated and
untreated primary sludge until around day 11. The total
duration of the BMP tests was enough to convert the
hydrolyzed substrate to biogas as seen by a plateau in the
Fig. 4. Subsequent conversion of the optimal wild-type enzyme
cocktail Protease : Lipase ratio of 1:3 (Enzyme treated) and
primary sludge (Control) mix through Biomethane potential
(BMP) test. Points, methane production; Solid lines, non-linear
curve fit for the treated and untreated primary sludge for the
determination of methane production rates (k) for the first 10 days
of BMP test.
Enhancing Hydrolysis of Sludge by Enzyme Mixture
methane production starting from around day 12. At this
time, the microorganisms in the reactors are utilizing
almost all the remaining degraded organics in the system.
The enzyme-treated primary sludge exhibited a higher
methane production rate (i.e., k = 2.2/d) compared to the
untreated primary sludge (i.e., k = 1.9/d). These k values
show that the enzyme-treated primary sludge was faster in
terms of methane production relative to the untreated
primary sludge. The observed increase in biogas production
and methane yield was obtained by comparing the enzyme-
treated and untreated primary sludge. The enzyme-treated
sludge clearly showed an increase in biogas production and
methane yield because the organics present in the sludge
undergone complete degradation comparative to the
untreated sludge, thus leading to more efficient utilization
of microorganisms for bioconversion [43,44]. The higher
biogas production and methane production rate is due to
the more efficient hydrolysis of lipids and proteins in the
enzyme-treated primary sludge. The enzyme hydrolyzed
primary sludge was a better substrate because the components
were made up of simpler organic compounds compared to
the polymeric organics still present in the untreated primary
sludge. The simpler organic compounds were readily
available to the microorganisms as substrates leading to
faster and more efficient bioconversion to biogas.
4. Conclusion
Lipase and protease were isolated from wild-type cultures
harboring enzyme-producing microorganisms from digesters
treating secondary sludge. Enzymatic treatment using wild-
type protease and lipase was found to be effective in
degrading the organics present in primary sludge. VSS
reduction was used as a basis to measure this effectiveness
and it was found that the ratio P:L = 1:3 was optimal in
degrading primary sludge components. A VSS reduction of
33.3% was observed in this manner. BMP tests showed
that enzymatically treated primary sludge increases biogas
production by 84.1%, methane production by 89.8%, and
methane yield by 9.6%. An increase in methane production
rate was also observed from the treated primary sludge.
Enzymatic treatment of sludge is gaining traction nowadays
and it will be a promising trend in the future. Other enzymes
should also be tested and optimized.
Acknowledgements
This work was financially supported by the Korea Institute
of Energy Technology Evaluation and Planning, Republic
of Korea (KETEP) and the Ministry of Trade, Industry &
139
Energy (MOTIE) of the Republic of Korea (No.
20163030091540). This work was also supported by the
‘Human Resources Program in Energy Technology’ of the
KETEP Grant, funded by MOTIE, Republic of Korea (No.
20144030200460). This work was financially supported
by the National Research Foundation of Korea (NRF)
grant funded by the Korea government (MSIT) (No.
2018R1A2B3005232).
The authors declare no conflict of interest.
Neither ethical approval nor informed consent was
required for this study.
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