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Enzyme research and applications in biotechnological intensification of biogas

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Article  in  Critical Reviews in Biotechnology · August 2011

DOI: 10.3109/07388551.2011.595384 · Source: PubMed

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 Critical Reviews in Biotechnology, 2011, 1–15, Early Online
© 2011 Informa Healthcare USA, Inc.
ISSN 0738-8551 print/ISSN 1549-7801 online
DOI: 10.3109/07388551.2011.595384

REVIEW ARTICLE

Enzyme research and applications in biotechnological

intensification of biogas production
Wilson Parawira

Department of Applied Biology, Kigali Institute of Science and Technology (KIST), Avenue de I’ Armee, B.P. 3900 Kigali,
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11

Rwanda

Abstract
Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local
resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas),
and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic
materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic
solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production
of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of
pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the
most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve
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biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into
fermentable structures. A number of authors have reported significant improvement in biogas production when
crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on
the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of
energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as
oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the
food processing industries and have been considered very difficult to convert into biogas. Improved methane yield
has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing
microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been
shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme
dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated.
This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of
complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass
are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic
hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering
are addressing this issue.
Keywords:  Enzymes, hydrolysis, complex organic matter, anaerobic digestion, improved biogas production

Introduction complex sequential, and parallel biological reactions in

Anaerobic digestion of energy crops, residues, and
wastes is of increasing interest in order to reduce green-
house gas emissions and to facilitate a sustainable
development of energy supply. Production of biogas
provides a versatile carrier of renewable energy, as
methane can be used for replacement of fossil fuels in
both heat and power generation and as a vehicle fuel.
Anaerobic digestion ­consists of several interdependent,
the absence of ­oxygen, during which the products from
one group of microorganisms serve as the substrates
for the next, resulting in ­transformation of organic mat-
ter mainly into a mixture of ­methane and carbon diox-
ide (Parawira, 2004; Noykova et  al., 2002; Pavlostathis
and Giraldo-Gomez, 1991; Gujer and Zehnder, 1983).
A ­simplified anaerobic digestion ­process is shown in
Figure 1. Complex ­polymers are ­broken down to soluble

Address for Correspondence:  Parawira Wilson, Department of Applied Biology, Kigali Institute of Science and Technology (KIST), Avenue de
I’ Armee, B.P. 3900 Kigali, Rwanda. Email: parawiradr@yahoo.co.uk
(Received 19 November 2010; revised 17 May 2011; accepted 06 June 2011)

1
 2  Parawira Wilson
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Figure 1.  A simplified anaerobic digestion process.
products by enzymes produced by fermentative bacteria
(Figure 1, Group 1) which ­ferment the substrate to short-
chain fatty acids, hydrogen, and carbon dioxide. Fatty
acids longer than acetate are metabolized to acetate
by obligate hydrogen-producing acetogenic bacteria
(Figure 1, Group 2). The major products after digestion of
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the substrate by these two groups are hydrogen, carbon
dioxide, and acetate. Hydrogen and carbon dioxide can
be converted to acetate by hydrogen-oxidizing ­acetogens
(Figure 1, Group 3) or methane by carbon-dioxide-
­reducing, hydrogen-oxidizing ­methanogens (Figure 1,
Group 4). Acetate is also converted to methane by aceti-
clastic methanogens (Figure 1, Group 5). Nearly seventy
per cent of methane from biogas digesters is derived from
acetate. Materials not converted together with microbial
biomass accumulate as a residue or sludge that can be
used as fertiliser in crop production. The digestate from
anaerobic fermentation is a valuable fertilizer due to the
increased availability of nitrogen and the better short-
term fertilization effect. Methane and carbon dioxide
are the principal end products, with minor quantities
of nitrogen, hydrogen, ammonia, and hydrogen sulfide.
Sources that generate biogas are numerous and varied
and include landfill sites, wastewater treatment plants,
and anaerobic digesters. Biogas can be produced from a
variety of biodegradable waste feedstocks including sew-
age sludge, municipal waste, food industry wastewaters,
agricultural residues and energy crops. The vast amounts
of municipal, industrial, and agricultural wastes that
are released every day, in every country, create serious
­environmental problems.
Polymeric carbohydrates, lipids and proteins ­present
in particulate organic matter cannot be taken up by
microbial cells. Therefore, microorganisms produce
and excrete hydrolytic enzymes such as amylases,
cellulases, proteases, and lipases to breakdown and
­solubilize the macromolecular structures into soluble
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matter such as simple sugars, amino acids, glycerol and
fatty acids to facilitate transport through the cell mem-
brane (Mshandete et  al., 2007). Once inside the cell,
these simple molecules are used to provide energy and
to synthesize cellular components. Polysaccharides are
converted to simple sugars; hydrolysis of cellulose by the
cellulase enzyme complex yields glucose; hemicellulose
degradation results in monosaccharides such as xylose,
glucose, galactose, pentoses, arabinose, and mannose,
while starch is converted to glucose by amylase enzymes.
The hydrolysis is normally rate-limiting if the substrate
is in particulate form, and especially the lignocellulose-
rich matter. This requires an understanding of hydrolytic
enzyme production and activities in the hydrolytic/­
acidogenic phase of the anaerobic digestion (Zhang
et al., 2007; Parawira et al., 2005).
A complex consortium of microorganisms ­participates
in the hydrolysis and fermentation of organic mate-
rial. Microbial diversity in biogas digesters is as great as
that of rumen wherein seventeen fermentative bacte-
rial ­species have been reported to play an important
role for ­production  of biogas. Bacteria excrete enzymes
that ­hydrolyse the particulate substrate to small trans-
portable molecules, which can pass through the cell
­membrane. Most of the bacteria are strict anaerobes
such as Bacteriocides, Clostridia and Bifidobacteria.
In addition, some facultative anaerobes such as
Streptococci and Enterobactericeae take part (Weiland,
2010). Furthermore, it is the nature of the substrate
that determines the type and extent of the fermenta-
tive bacteria present in the digester (Parawira et  al.,
2005). Parawira et  al. (2005) reported higher amylase
activity during mesophilic anaerobic digestion of solid
potato waste compared to other hydrolases, suggesting
the presence of many amylolytic microbes. Ramasamy
et  al. (1990) reported a higher presence of proteolytic
organisms in cow dung-fed digesters and other animal
Critical Reviews in Biotechnology
 Enzyme research and applications in biotechnological intensification of biogas production  3
waste-fed digesters. However,  Preeti Rao and Seenayya
(1994) observed that while cow dung-fed digesters sup-
ported higher amylolytic microorganism populations,
poultry waste-fed digesters showed higher proteolytic
populations. Among fermentative organisms, Bacteroides
succinogens, Butyrivibrio fibrisolvens, Clostridium cello-
bioparum, Ruminococcus albus and Clostridium sp. were
predominant. Cellulolytic Clostridia have been reported
to be important in anaerobic environments rich in
plant materials where they are responsible for recycling
­cellulose. They are also dominant in anaerobic digest-
ers fed with municipal solid waste or agricultural raw
materials ­containing a high percentage of lignocellulosic
compounds (Guedon et  al., 2002). During the last few
decades, anaerobic digestion of organic matter has been
presented as a suitable technology used for the treat-
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ment of organic wastes and production of energy from
the combustion of biogas (Lettinga, 2001; Lema et  al.,
2001; Parawira, 2004). Anaerobic digestion technology
has evolved quickly and, at present, can be competitive
with aerobic systems, especially for treating industrial
wastewater and organic solid waste with high chemical
oxygen demand.
Studies on the application of enzymes to
pretreat complex organic matter
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Anaerobic digestion of lignocellulosic material
Lignocellulosic materials are often a major or some-
times the sole components of different waste streams
from ­various industries, forestry, agriculture, and
­municipalities. In the rural areas of most countries
various ­cellulosic ­biomass (cattle dung, agricultural
residues, etc) are available in abundance. The ligno-
cellulosic materials represent the largest renewable
­reservoir of potentially fermentable carbohydrates
on earth. Lignocelluloses comprise a large fraction of
municipal solid waste, crop residues, animal manures,
woodlot ­arisings, ­forest ­residues or dedicated energy
crops. Anaerobic digestion of waste ­lignocellulosic feed-
stocks for biogas ­production is a ­biological process that
combines renewable energy generation with sustain-
able waste treatment. Lignocellulose is the ­complex and
rigid matrix of plant cells and is composed of cellulose,
hemicelluloses, lignin, extractives, and several inorganic
materials (Taherzadeh and Karimi, 2008). In the gen-
eration of biogas from agricultural wastes, cellulose and
hemicellulose components are converted into methane
and carbon dioxide. Lignin can however, not be degraded
under anaerobic conditions (Fernandes et al., 2009). The
rate limiting step for anaerobic digestion of lignocellu-
losic material is the hydrolysis of cellulose and hemicel-
luloses into mono-, di- and oligosaccharides. Increasing
the hydrolysis rate is critical in order to improve the
biomass-conversion efficiency of anaerobic digestion
(Bruni et  al., 2010). Biological degradation of lignocel-
lulosic material is normally facilitated by enzymes, such
as cellulases and hemicelluloses, which are produced by
© 2011 Informa Healthcare USA, Inc.
the microorganisms in the digester. However, enzymatic
hydrolysis of lignocelluloses with no pretreatment is
­usually not so effective because of the high stability of the
materials to enzymatic attacks. They are resistant to enzy-
matic attack because of the tight association between
lignin, cellulose, and hemicelluloses. The crystallinity of
cellulose, its accessible surface area and protection by
lignin and hemicelluloses, degree of cellulose polym-
erization, and degree of acetylation of hemicelluloses
are the main factors considered as affecting the rate of
biological pretreatment of lignocelluloses by enzymes.
In full-scale biogas plants digesting lignocellulosic mate-
rial, the low digestibility of the biofibers contained in the
material causes loss of methane production and limits
the overall efficiency of the process (Jin et  al., 2009).
Therefore, pretreatment methods facilitating the accessi-
bility of holocellulose (cellulose and hemicelluloses) are
needed to increase the biogas potential of lignocellulosic
material.
Many pretreatment methods for increasing the biode-
gradability of lignocellulosic material have been reported
(Demirbas, 2008). Pretreatment methods include milling,
irradiation, steam explosion, ammonia fiber explosion,
supercritical CO2 and its explosion, alkaline hydrolysis,
liquid hot-water pretreatment, organosolv processes,
wet oxidation, ozonolysis, dilute- and concentrated-
acid hydrolysis. These various pretreatment methods
result in improvement in destruction of the crystallinity,
improvement in the accessible area, and destruction of
the ­protection by lignin and hemicelluloses, thereby
enhancing the bio-digestibility of the wastes for biogas
or ethanol production. However, most of these physical
and ­chemical pretreatments have been found to ­produce
inhibitory compounds to the fermenting yeast in ­ethanol
­production. Many strategies are being developed to
reduce the effect of the ­lignocellulosic inhibitors for
improved ethanol production (Parawira and Tekere,
2010). There are no similar reports on the effect of the
physical and chemical pretreatments by-products on
biogas ­production. Therefore, there is need to ­investigate
the effect of by-products from these ­physical-chemical
pretreatment methods on the consortium of micro-
organisms involved in biogas production, if they are
to be adopted to improve biogas production from
­lignocellulosic material.
Biological pretreatment using microorganisms can
also be used to treat the lignocelluloses and enhance
anaerobic digestion processes (Hendriks and Zeeman,
2009; Yadvika et al., 2004). Use of vital microorganisms is
probably more dynamic and efficient than free enzymes
due to their ability of regeneration and concomitant
production of diverse enzyme responding to the given
substrate. The applied microorganisms usually degrade
lignin and hemicelluloses but only a very little part of the
cellulose, since cellulose is more resistant than other parts
of lignocelluloses to the microbial attack (Taherzadeh
and Karimi, 2008). Several fungi e.g., brown-, white- and
soft-rot fungi have been used for the purpose of making
BBTN 595384
 4  Parawira Wilson
the cellulose and hemicelluloses more accessible for
further enzymatic hydrolysis. Microorganisms capable of
converting polysaccharides to monosaccharides include
cellulase-producing Trichoderma reesei and Trichoderma
viride. Srilatha et al. (1995) obtained a 33% methane yield
increase from orange processing waste by treatment with
selected fungi strains. Similarly, Taherzadeh and Karimi
(2008) reported improved enzymatic hydrolysis (94%
sugar recovery) when treating office paper with selected
aerobic bacteria. An edible mushroom, Pleurotus sajor-
caju, which is cultivated on a variety of agricultural
residues, possesses the capacity to degrade cellulose,
hemicelluloses and lignin components (Bisaria et  al.,
1983). Biogas can then be produced from the degraded
agricultural wastes. Muller and Trosch (1986) reported
twice as much biogas yield from fungus (Pleurotus sp.
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florida) treated straw than from untreated straw. They
had grown twenty two basidiomycetes, mostly white
rot fungi on wheat straw and found that this oyster
mushroom showed fastest delignification of the straw.
Their results showed that “myco-straw” can be better
hydrolysed and converted to biogas in comparison to
untreated straw. After biological lignin removal the straw
cellulose was better accessible for anaerobic digestion.
The procedure involving microbial delignification and
biogas ­production, offers the possibility of utilizing and
­removing the waste wheat straw in a completely bio-
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logical way. The useful products from this process are
­mushroom in the first step and methane in the second
step. Geeta et  al. (1994) found that sugarcane bagasse
pretreated with Phanerochaete chrysosporium for 3 weeks
under ambient temperature conditions produced higher
biogas compared with cattle excreta. Phanerochaete
chrysosporium is the most investigated fungus for lignin
degradation. Akao et  al. (1992) reported enhanced
anaerobic digestion of citrus peels with an enzyme
solution from Aspergillus sp. A-1. The enzyme solution
was reported to have cellulase and ­pectinase activities
that allowed the anaerobic digester to be ­operated at a
higher organic loading rate. Biological ensilage ­additives
with hetero- and homo-fermentative activity (Silasil
energy®) as well as enzymes (Sil-all 4 x4®) or bacteria
and yeasts (Microferm®) ­effectively increased the biogas
production per organic dry ­matter of maize (11.8, 10.1,
and 14.7%, respectively) after 7  weeks of pretreatment
(Vervaeren et al., 2010). However, treatment rate is very
low in most biological pretreatment processes. At present
the process of decomposing ­cellulose and other complex
molecules into simple sugars and other compounds
relies on naturally occurring bacteria. The efficiency of
cellulose breakdown could be improved by using ­better
strains, particularly for cellulolysis, the limiting step of
the process. Guedon et  al. (2002) demonstrated that
fermentation of cellulose, the most abundant and renew-
able polymer on earth, can be greatly improved by using
a genetically engineered Clostridium cellulolyticum
with improved cellulolytic properties. Bagi et  al. (2007)
­demonstrated an increase of biogas production by about
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60–70% due to inoculation of biogas reactors of up to
5m3 size with external hydrogen-producing bacteria with
­cellulolytic activity (Caldicellulosyruptor ­saccharolyticus)
in long-term experiments. Cellulolytic strains of ­bacteria
like Actinomycetes and mixed ­consortia have been found
to improve biogas production in the range of 8.4–44%
from cattle dung (Tirumale and Nand, 1994; Attar
et  al., 1998). All the strains ­exhibited a range of ­activity
of all the enzymes involved in ­cellulose ­degradation,
namely C1 enzyme, exoglucanase, ­endoglucanase,
ß-glucosidase. Weib et  al. (2010) reported enhance-
ment of biogas ­production by addition of a consor-
tium of hemicellulolytic bacteria (Bacteroides species,
Azospira oryzae and diverse species within the order of
Clostridiales) immobilized on activated zeolite. Lissens
et al. (2004) reported advanced anaerobic bioconversion
of lignocellulosic waste for bioregenerative life support
following thermal water treatment and biodegradation
by Fibrobacter ­succinogenes. The bacterium, Fibrobacter
succinogenes is widely considered as one of the most
active and most important cellulose-digesting anaerobic
bacteria in the rumen.
There have been some studies on the use of enzymes
to pretreat some lignocelluloses for improvement of
biogas production. Sonakya et  al. (2001) pretreated
wheat grains with Trizyme (cellulose, α-amylase and
protease) prior to anaerobic digestion and observed
an increase in methane production by 7–14%. These
­studies suggest that the addition of exogenous enzymes
can improve the performance of anaerobic digestion
­systems. However, enzyme activity can be affected by
many factors ­including the substrate, incubation time,
system configuration, and environmental conditions
(e.g., temperature and pH). More research is needed to
determine if and when the addition of enzymes such as
cellulases and hemicellulases to the anaerobic diges-
tion system will improve digestion rates and biogas
yields of lignocellulosic biomass. For example, enzymes
could be added into a single-stage anaerobic digester or
could be used to pre-treat the biomass material prior to
anaerobic digestion. In two-stage anaerobic digestion,
enzymes could be added to the hydrolysis stage prior to
biogasification. Romano et  al. (2009) addressed these
questions by investigating the effect of enzyme addi-
tion on Jose Tall Wheat Grass (wheat grass). Anaerobic
digestion systems were performed using batch reactors
operated at 50°C. The application of enzyme products in
three digestion configurations were simulated and inves-
tigated: (1) enzyme addition to a single-stage digester,
(2) pre-treatment of wheat grass with enzymes followed
by a ­single-stage anaerobic digestion and (3) enzyme
addition to the first stage (hydrolysis and acidification)
of a two-stage digestion system. The enzyme products
showed positive effects on the solubilization of wheat
grass when used alone to treat the wheat grass. However,
no significant difference in biogas and methane yields
and volatile ­solids ­reduction resulted when the enzyme
products were tested in the anaerobic digestion systems.
Critical Reviews in Biotechnology
 Enzyme research and applications in biotechnological intensification of biogas production  5
Romano  et  al. (2009) concluded that microorganisms
present in the natural inoculum in biogas reactors were
effective in carrying out the digestion of wheat grass.
However, there is need for further research on the effect
of adding enzymes to anaerobic reactors digesting ligno-
cellulosic material, given its potential as a source of raw
material for biogas production.
Anaerobic digestion of food waste
Wastewaters from the food processing industries have very
high concentrations of oil and grease, proteins and sug-
ars and therefore, high chemical oxygen demand levels,
which are difficult to treat through conventional ­biological
treatment systems. However, the oily wastewaters could
be cheap and abundant sources of biogas. Lipids, char-
acterised as fats or oils and greases, are one of the major
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organic matters found in food wastes and some industrial
wastewaters, such as those from slaughterhouses, edible
oil processing industry, restaurant waste, dairy indus-
tries, fish industry or fat refineries, and sewage sludge
(Gannoun et al., 2009; Luste et al., 2009; Fernandez et al.,
2005; Perle et al., 1995; Cirne et al., 2006). Lipids included
in food waste and wastewater consists mainly of triacylg-
lycerides and long-chain fatty acids (LCFA). In anaerobic
treatment systems, triacylglycerides can be hydrolysed
by extracellular lipases to LCFA and glycerol. Glycerol is
converted to acetate by acidogenesis while LCFA are suc-
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cessively degraded via ß-oxidation pathway to acetate
and hydrogen, which in turn are converted to methane.
If compared with other organic matter like carbohydrates
and proteins, lipids are attractive for biogas production
due to the fact that they are reduced materials and have
a high theoretical methane potential which can contrib-
ute to increased biogas production and consequently
improves the plant economy (Salminen and Rintala, 2002;
Pereira et al., 2003; Kim et al., 2004). Theoretically 1.01 L of
methane at standard temperature and pressure (STP) can
be produced from for example 1 g of oleate (unsaturated
LCFA, C18:1), whereas only 0.37 L can be produced from
1 g of glucose (Cavaleiro et al., 2008). In this context, lipid-
rich waste can be regarded as a large potential renewable
energy source. However, anaerobic treatment of organic
wastes with high lipid and protein content present sev-
eral operational problems which limit the potential to
obtain methane from them (Palatsi et  al., 2009; Cirne
et al., 2007). Fats and proteins present in these effluents
have a low biodegradability coefficient (Cammarota et al.,
2001; Valladao et al., 2007). The fats may solidify at lower
temperatures and cause operational problems such as
clogging and developing unpleasant odors, represent-
ing a serious problem of anaerobic digestion processes.
Liquefaction of lipids was found to be rate-limiting in
slaughterhouse wastewater when high amounts of sus-
pended solids were present due to their low bioavailabil-
ity (Sayed et al., 1988). Petruy and Lettinga (1997) found
similar results when treating a milk-fat emulsion in an
expanded granular sludge bed reactor equipped with
a sieve-drum at the top of the reactor to prevent floated
© 2011 Informa Healthcare USA, Inc.
sludge from being washed out. Adsorption of lipids onto
biomass can cause sludge flotation and washout (Amaral
et al., 2004; Perle et al., 1995). Furthermore, oil and grease
adsorbed on the surface of the anaerobic sludge may
limit the transport of soluble substrates to the biomass
and consequently reduce the rate of substrate conversion
(Pereira et al., 2004; 2005). It has also been reported that
high concentrations of LCFA can destabilize anaerobic
digesters due to inhibition of methanogenic bacteria
by possible damage to the cellular membrane (Mendes
et  al., 2006; Hanaki et  al., 1981, Angelidaki and Ahring,
1992). Hanaki et al. (1981) conducted an extensive study
of the inhibitory effects of LCFA on anaerobic digestion.
They concluded that LCFAs affect obligate hydrogen-
producing acetogenic bacteria, which are responsible
for the ß-oxidation of LCFA as well as hydrogentrophic
and acetotrophic methanogenic archaea, which convert
the intermediates resulting from the ß-oxidation of LCFA
process. Different values of inhibition concentration for
different LCFA are reported, for example concentrations
of inhibition are in the range of 30–300 mg1l–1 for oleic
acid, 100–300 mg1l–1 for stearic acid and 30 mg1l–1 for
linoleic acid (Fernandez et al., 2005).
Different pretreatment methods have been investi-
gated to address the problems caused by oily wastewa-
ter and improve the digestion process. These methods
include removing the oil and grease by using ­equipment
like grease boxes; oil/water separators and flotation
systems; chemical pretreatment of the waste by NaOH,
Ca(OH)2 or HCl; and biological pretreatment using
enzymes or lipolytic microorganisms. However, each of
these methods has its own drawbacks. When using grease
boxes, ­dissolved and/or emulsified oil and grease are
not retained in these units and enter into the anaerobic
­treatment systems, causing considerable problems. Masse
and coworkers (2001) did not recommend ­pretreatment
with an alkali because it results in an increase in pH in the
digestion process. Among these strategies, the alternative
of using specific enzymes, lipases, has recently gained
more attention because of stringent environmental regu-
lations and clean and friendly application of enzymes
(Cammarota et al., 2001; Jeganathan et al., 2007; Mendes
et  al., 2005). Adding enzymes into anaerobic digesters
treating food-processing wastes have been reported to
result in improved digestion and biogas production.
Lipases have shown to be a very promising alternative
for degrading rich-lipids wastewaters generated by dairy
and slaughterhouses industries (Leal et al., 2006; Mendes
et al., 2006).
However, a chief obstacle preventing more studies and
widespread use of enzymes in environmental technology
is the high cost of commercial enzymes. Consequently, the
employment of hybrid technology (enzymatic treatment
combined with biological treatment) using ­enzymatic
pools produced through solid-state ­fermentation from
low-cost industrial wastes may represent an important
advancement in the treatment of wastewaters with
high oil and grease contents (Cammarota and Fereire,
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 6  Parawira Wilson
2006; Leal et  al., 2006). Mendes et  al. (2006) investi-
gated the effect of enzymatic hydrolysis pretreatment
of lipids-rich dairy wastewater on anaerobic digestion.
A low-cost commercially available lipase preparation
from an animal source was used to perform enzymatic
hydrolysis pretreatment of lipid-rich wastewater from
dairy industry which resulted in increased levels of bio-
gas production and higher organic (COD) removal. The
pretreatment was optimized for a 12 h hydrolysis time
enabling high biogas formation and organic removal.
Promising results in relation to the biogas formation were
also found when the hydrolysis and biodegradation steps
were carried out simultaneously. Another ­application
of low-cost enzymes for pretreatment of effluents from
poultry slaughterhouses was reported by Valladao et al.
(2007). A pool of hydrolases with 21.4 U g–1 lipase activ-
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ity was produced through solid-state fermentation of the
fungus Penicillium restrictum in waste from the Orbignya
oleifera (babassu) oil processing industry. Enzymatic
hydrolysis and anaerobic biodegradability tests were
conducted on poultry slaughterhouse effluents with
increased oil and grease contents (150–1200 mg l–1) and
solid enzymatic pool concentration (0.1–1.0%) w/v).
Enhanced anaerobic treatment efficiency relative to raw
effluent (COD removal efficiency of 85% versus 53% and
biogas production of 175ml versus 37 ml after 4 days) was
achieved when a 0.1% concentration of enzymatic pool
For personal use only.
was used in the pretreatment stage with 1200 mg oil and
grease l–1. Cammarota et  al. (2001) reported that enzy-
matic pretreatment using an enzymatic cocktail of lipases
resulted in improvement of anaerobic degradation in an
UASB reactor of dairy wastewater containing elevated fat
levels compared with untreated wastewater. The dairy
wastewater was pre-treated with 0.1% (w/v) of fermented
babassu cake containing Penicillium restrictum lipases
obtained from solid state fermentation. The application
of low cost lipases as a pretreatment to hydrolyze and
dissolve lipids may improve the anaerobic degradation
of wastewaters rich in lipids, enhancing the production
of biogas. With slaughterhouse wastewater, pancreatic
lipase PL 250 (4–24h) reduced the average particle size
by 60% and increased lipid hydrolysis by 40% during 24h
(Mendes et al., 2006). Luste et al. (2009) investigated the
use of a biological product containing hydrolytic enzymes
during solubilization of organic material, i.e., hydrolysis
and methane production potentials on ­different by-
products from meat-processing plants. The biological
product Liquid Certizyme 5TM increased soluble COD
of digestive tract and drumsieve waste the most as com-
pared to untreated material, (62% and 96%, respectively).
However, methane production potential was decreased
compared to untreated materials apparently due to
inhibition by hydrolysis products such as VFA from
acidification of solubilized products. Masse et al. (2001a)
observed that 35% of the natural fats in slaughterhouse
effluents hydrolysed to free LCFA when treated with pan-
creatic lipase 250 (PL-250), increasing the rate of removal
in an anaerobic reactor operating in batch conditions.
BBTN 595384
The pretreatment had a significant effect on reduction of
the particle size, but only a small effect on their digest-
ibility, which was only 4% higher than that of the control
substrate after 69-hour digestion. The authors concluded
that this type of pretreatment can be more efficient in
reactors that operate at high temperatures, since the rate
of LCFAs removal can be increased (Masse et al., 2001a).
Rintala and Ahring (1994) investigated addition of
Pulpzyme HA (xylanase and cellulase), Alcalase 2.5LB
(protease), and Resinase A 2X (lipase) to the anaerobic
digestion of source-separated household solid waste.
In batch treatments, enzymes were added individually
or as a mixture, and the specific methane activity was
measured (defined as the slope of the cumulative meth-
ane production from the initial 20–30 h of digestion per
volatile solids (VS) content of the inoculum). Only the
lowest concentration of protease applied (0.5 ml/kg VS)
resulted in significantly higher (11%) specific methane
activity. However, the authors reported no significant
differences in biogas yield or VFA concentration in the
reactor effluent between enzyme-treated and non-
enzyme treated continuous digester systems. Studies on
enzymatic pretreatment of slaughterhouse wastewater
using commercial lipase from animal, microbial, and
vegetable sources did not also significantly enhance the
anaerobic digestion process (Masse et al., 2001a; Masse
et al., 2003). In addition, Masse et al. (2003) concluded
that the use of commercial enzymes for direct enzymatic
bioaugmentation makes the anaerobic digestion pro-
cess quite ­expensive and thus not economically feasible.
The possibility of pretreatment with enzyme-producing
aerobic microorganisms was demonstrated with a
lipolytic fungus by Cammarota et  al. (2001) and with
mixed bacterial cultures comprising lipase, protease and
amylase producers (Mongkolthanaruk and Dharmsthiti,
2002). However, when using aerobic microorganisms for
pretreatment, oxygen supply may be required thereby
increasing the cost of the process. As an alternative,
addition of anaerobic microorganisms as a means of
improving xylanolytic, cellulolytic, hemicellulolytic, and
lipolytic activities has been investigated (Angelidaki and
Ahring, 2000, Mladenovska et  al., 2001). In a study by
Cirne (2006), it was reported that direct lipase enzyme
addition and bioaugmentation of anaerobic digester
with a lipolytic microbial strain (Clostridium lundense
(DSM 17049T) enhanced lipid hydrolysis resulting in
improved biogas production rate and accordingly, a
reduction in the digestion period required in obtain-
ing the same methane yield as the control. Gumisiriza
et  al. (2009) reported enhanced biogas production
(68%) after pretreatment of fish ­processing wastewater
with unidentified bacterial strains isolated from a local
stabilization pond treating high strength fish processing
wastewater. Bioaugmentation offers the possibility of
enzyme ­production over a long period of time provided
that the ­microorganism added is able to compete with
the other microbes present in the reactor. However, the
other major drawback of using bioaugmentation under
Critical Reviews in Biotechnology
 Enzyme research and applications in biotechnological intensification of biogas production  7
a­ naerobic conditions is the accumulation of LCFAs,
which inhibit the production of biogas. In a previous
study on the influence of lipid concentrations on hydro-
lysis and biomethanisation of lipid-rich waste by Cirne
et al. (2007), the ­addition of a commercial lipase improved
lipid hydrolysis. In the same study, the inhibition of
methane production caused by LCFAs ­accumulation
was found to be more significant than the inhibition due
to volatile fatty acids ­accumulation. Lipid-containing
waste has very high methane production potential and
therefore research on the limiting steps of the conver-
sion of lipids to methane need to be continued.
There are several research studies available on the
treatment of oily wastes with artificially added fats using
free lipase (Cammarota et al., 2001; Masse et al., 2001 and
2003; Mongkolthanaruk and Dharmsthiti, 2002). There is
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
little information available on oily wastewater treatment
using immobilized lipase. Free lipases are generally
­soluble and unstable, hence can be used once in ­solutions.
In addition, free lipase is not only often inactivated due
to different environmental conditions (ionic strength,
pH, inhibitors) but also too expensive to utilize in waste-
water treatment. To overcome these problems lipase
can be immobilized on a suitable media. Immobilized
lipase has the advantages of multiple usage, controlled
reactions and thermostability (Matsumoto and Ohashi,
2003). In addition, for continuous operation in packed
For personal use only.
bed reactors or fluidized bed reactors, immobilized
lipases yield higher volumetric productivity ­compared
to free lipases (Jeganathan et al., 2007). Jeganathan et al.
(2007) ­evaluated the hydrolysis of wastewater with high
oil and grease concentration from the pet food ­industry
using immobilized lipase as a pretreatment step for
anaerobic treatment through batch and continuous-flow
experiments.
Anaerobic digestion of sewage sludge
The volume of wastewater sludge produced annually from
treatment of the various municipal and industrial waste-
waters is very high in many countries and poses serious
and complex disposal problems (Cui and Jahng, 2006).
Digested sludge is a complex material ­constituted by
­particles, bacteria, and extracellular ­polymeric ­substances
that are excreted by bacteria. Sludges ­produced from
­primary and secondary settling tanks as a result of
­aerobic and anaerobic digestion ­processes typically have
a solid content of 0.5–5% (Parmar et al., 2001). This solid
­component of sludge consists of organic and inorganic
material in an approximate ratio of 60:40; hence sewage
sludge can be used for biogas production. Generally, only
approximately half of the organic matter in waste ­activated
sludge is susceptible to direct biodegradation by anaero-
bic digestion resulting in the ­formation of biogas (Gossett
and Belser, 1982). The remaining non-digestible material
is either inorganically bound carbon or slowly digestible
organics. Bacterial biomass is a major solid component
of sludge biomass and is developed ­during the sewage
treatment processes. Most of the organic components
© 2011 Informa Healthcare USA, Inc.
present in sludge biomass are located within microbial
cells ­generated during the activated sludge process. The
cell wall of microorganisms is a stable semi-rigid structure,
consisting of glycan strands cross-linked by peptide chains.
This cell wall imposes resistance to biodegradation leading
to long hydraulic retention times of the order of 20–30 days
for the biological stabilisation of sewage sludge (Weemaes
and Verstraete, 1998; Tanaka et al., 1997). In addition, there
is also a fraction of lignocellulosic materials in sludge,
which are also complex in structure. Disintegration of
sludge biomass by chemical, mechanical or biological
forces is therefore required to destroy floc structures and
disrupt cells to release the ­soluble substances and fine
particles. Eastman and Ferguson (1981) reported that
the rate-­limiting step ­during anaerobic digestion was the
hydrolysis of waste from activated sludge. The ideal way
to solve the sludge associated ­problems is to enhance the
hydrolysis of the sludge biomass into fermentable struc-
tures that can be used for ­production of biogas or other
valuable chemicals. Each ton of sludge biomass that is
converted will of course result in biogas, and it also repre-
sents a substantial ­saving on the handling of the residual
sludge. For example, it is possible to obtain methane yields
of approximately 400 m3 CH4 per ton of volatile solids from
enzymatically treated digested mixed primary and waste
activated sludge (Davidson et al., 2007).
Pretreatment processes involving mechanical,
chemical, and physical methods have been developed
to improve the sludge disintegration rate. Enzymatic
hydrolysis of sludge biomass is an environmentally
friendly alternative to mechanical, chemical and/or
heat treatment technologies for converting sludge into
fermentable structures. The fermentable structures from
the sludge can then be converted into value added prod-
ucts such as biogas. Enzymatic hydrolysis of sludge is
mild, quick, simple to implement and more specific in
its action and therefore efficient as a pre-treatment for
sludge biomass. The efficiency of the enzymatic process
is quite high and the mild process conditions require
neither expensive materials nor high process energy
compared to mechanical and chemical breakdown. The
arguments in favor of enzymes to pretreat sludge ­biomass
are compelling. A number of authors have reported a
significant reduction in sludge volume and improved
efficiency of wastewater treatment processes when com-
mercial enzymes are added. Enzymes act on ­specific
substances present in municipal sludge converting the
complex material into simple utilisable monomers.
Considerable research has been conducted during the
past two decades to investigate the possibilities offered
by enzymes in improving sludge biomass treatment.
Some of the reasons for this interest include the growing
recognition that biotechnological advances are allowing
the production of cheaper and more readily available
enzymes through genetic engineering of microorgan-
isms, and better isolation procedures (Karam and Nicell,
1997; Gerhardt et al., 2007; Schimpf and Valbuena, 2009;
Weiland, 2010).
BBTN 595384
 8  Parawira Wilson
Enzyme treatment of wastewater sludge biomass
A large number of different enzymes have been reported
to play an important role in a range of sludge treatment
applications. Enzyme treatment can potentially acceler-
ate the solubilization of the sludge and thus minimise
the rate-limiting step in the digestion processes involved
(Tiehm et  al., 2001; Watson et  al., 2004; Whiteley et  al.,
2002). The solubilization of solids is achieved by two
groups of enzymes: the lytic enzymes resulting in cell
wall disruption and the hydrolytic enzymes causing the
breakdown of macromolecules (Mayhew et al., 2002).
Sludge biomass can be converted into biogas through
enzymatic hydrolysis and anaerobic digestion done
simultaneously or sequentially (Figure 2). Efficient
hydrolysis of sludge biomass requires a number of
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
enzymes due to its multiple components. The enzymatic
pre-treatment of mixed sludge by either endogenous or
added industrial enzymes, prior to anaerobic digestion
processes, has been shown to result in improved degra-
dation of the sludge and to improved biogas production
as shown in Table 1. The enzymatic treatment of mixed
sludge by added enzymes prior to anaerobic digestion
has been shown to result in improved ­degradation of
the sludge and increase in methane production. The
­addition of cellulase (Celluclast 200L) and ß-glucosidase
For personal use only.
Figure 2.  Generalised scheme of sludge biomass to biogas conversion process.
BBTN 595384
(Novozyme 188) to anaerobic digesters treating ­sewage
sludge was reported by Higgins and Swartzbaugh (1986)
to result in increased biogas production. The system they
used consisted of an enzyme pretreatment stage followed
by anaerobic digestion and resulted in increased biogas
and methane yields of 12% and 15%, respectively.
The effect of microbial enzymes in enhancement of
anaerobic digestion (60%) by enzyme addition in batch
digestion tests of sewage sludge was demonstrated by
Davidsson et al. (2007). A pre-hydrolysis step with addi-
tion of enzymes at controlled temperature over 4 hours
was shown to increase the methane yield significantly.
Further tests with direct feeding of enzymes together
with fresh sludge to the digester gave the same effect
of increase in methane yield. For implementation in
­full-scale, different strategies for adding the enzymes
were tested in both batch and pilot scale continuous
digestion. The addition of enzymes to the inlet together
with fresh sludge and addition with re-circulated
sludge were done. Both strategies resulted in significant
increases in methane production, although the former
gave a higher yield.
Wawrzynczyk (2007) also reported an improvement
in anaerobic digestion by the addition of two glycosidic
enzymes into an industrial-scale digester processing
Critical Reviews in Biotechnology
 Enzyme research and applications in biotechnological intensification of biogas production  9

Table 1.  Summary of work on hydrolysis of sludge using enzymes.

Sludge
Biological sludge (WWTP)
Primary & waste activated
sludge mixture (WWTP)
Mixed waste water sludge-
Anaerobically Digested sludge
(WWTP)
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
Scale Enzymes Effects Reference
Lab-scale Mixtures of lipase, Improved solubilisation of Wawrzynczyk
glycosidic enzymes organic matter (20–40%) et al., 2003
Reduced vitality 
Increased methane production
(60%)
Lab-scale Mixtures of enzymes Increased methane yield (60%) Davidsson
et al., 2007
Pilot-scale Mixtures of enzymes Increased methane yield (71%)
Full-scale Two glycosidic enzymes Increase methane Wawrzynczyk, 2007
production (20%)
Improved dewatering
properties
Lab-scale Protease, lipase, & other Enhanced dewaterability Ayol, 2005
(based on capillary suction
hydrolytic enzymes, time,

solid content of final product,

Primary sewage sludge (WWTP) Lab-scale Mixture of enzymes
(cellulase & pronase E)
Sewage sludge Lab-scale Cellulase, protease, lipase
Amylase, hemicellulase
filtrate turbidity)
Reduction of total Roman et al., 2006
solids (80%)
Improved solid reduction Parmar et al., 2001
and settling of solids

mixed wastewater sludge. The addition of the enzymes
resulted in improved biogas production and dewatering
properties of the digested sludge.
Roman et al. (2006) investigated the impact of addition
For personal use only.
by Lagerkvist and Chen (1993) in their study of a two-
step anaerobic degradation of municipal solid waste by
enzyme addition.
In continuous pilot-scale experiments, Davidsson

of cellulase and pronase E separately and in ­combination
on primary sludge and it was found that the mixture of
the two enzymes resulted in 80% ­reduction in ­solids
­compared to 20% in the control and 97% total COD
removal compared to 63% in the control. They concluded
that the enzyme addition at full scale could be expected to
lead to greater methane yields. However, primary sludge
is highly degradable and the results could be ­different
if more difficult to degrade waste activated sludge and
digested sludge which requires more enzyme treatment
were used.
Strategies for enzyme dosing to enhance anaerobic
digestion of sewage sludge
There also has been some research on the strategies for
enzyme dosing to enhance anaerobic digestion of sludge
biomass. In batch laboratory experiments by Davidsson
et  al. (2007) there was an increase in methane yield
for both sludge pretreated by enzymes (0.437 Nm3/kg
VSin) and for sludge in which there was direct feeding
of enzymes to the anaerobic digestion reactor (0.421
Nm3/kg VSin), compared with untreated sludge (0.348
Nm3/kg VSin). The enzymes added were divided into two
mixtures, mixture A and mixture B. Mixture A consisted
of four polysaccharide degrading enzymes and a lipase
and were immersed in an emulsifier combined with a
surface active substance. Mixture B contained protease
for complete hydrolysis of protein and glycoproteins
and was separately added 2 hours after mixture A had
been added to avoid hydrolysis of enzymes in mixture
A. They continuously adjusted the pH to 7 by addition
of sodium hydroxide. Similar results were also reported
et al. (2007) reported a higher methane yield (0.398 Nm3
CH4/kg VSin) achieved when enzymes were added directly
into the digester together with fresh sludge compared
with 0.366 Nm3 CH4/kg VSin when the enzyme was added
with digested sludge via a return pipe. A ­comparison
between methane potentials and methane yields showed
that 97–99% of the methane potential could be realised
in methane yield when enzymes were used. For the
untreated sludge 75% of the potential was realised in
continuous digestion.
Wawrzynczyk (2007) reported improved biogas pro-
duction and dewatering properties in a ­continuously
operating mesophilic full-scale wastewater ­treatment
plant during a six month period after addition of
two technical grade glycosidic enzymes supplied by
Novozymes A/S, Denmark. The dosage was 2.5 kg of each
enzyme solution per tonne feed dry solids to the diges-
tion chamber. The dosage point was the heat exchanger
system which ran every fourth hour for 30 to 40 ­minutes.
Preceding laboratory tests to determine the types of
enzymes and the dosage optimum had showed that the
two selected enzymes resulted in a better solubilisa-
tion of the sludge than proteases and lipases. The gas
­production from enzyme treated sludge increased by 10
to 20% in comparison to that from the reference digester
without enzyme. These results with studies done using
laboratory-, pilot- and full-scale reactors demonstrate
the potential to enhance biogas production from sewage
sludge using enzymes. Possible strategies for enzyme
dosing to enhance anaerobic digestion of sewage sludge
for biogas production have been attempted and the
results are encouraging.

© 2011 Informa Healthcare USA, Inc. BBTN 595384

 10  Parawira Wilson
Research on endogenous enzymes during
anaerobic digestion
Microbial consortia within anaerobic reactors mediate
the processes of polymer hydrolysis, fermentation to
organic acids and mineralization by methanogenesis.
There has been research into the endogenous enzymatic
activities during the hydrolysis stage to try to elucidate
the hydrolysis of complex polymers to monomers by
fermentative bacteria. An understanding of the endog-
enous enzyme activities in terms of their distribution
and relative activity in anaerobic digestion may result in
improvement biogas production from high-solid organic
matter. It is necessary to provide the right physical-
chemical ­environment to enhance the enzyme ­activities
in anaerobic reactors which may result in improved
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
­biogas production without adding extracellular enzymes.
Depending on the ­structure of the cell wall, the extracel-
lular enzymes have different locations. Some enzymes
are associated with the cell surface or released into the
bulk liquid, the enzymes may said to be cell-associated
or cell-free (Priest, 1984). The location of enzymes has
an impact on the degradation of the substrate (Parawira
et al., 2005). Two two-stage systems, one consisting of a
solid-bed reactor for hydrolysis/acidification connected
to an upflow anaerobic sludge blanket methanogenic
reactor, and the other consisting of a solid-bed reactor
For personal use only.
connected to a methanogenic ­reactor packed with wheat
straw ­biofilm carriers, were investigated with regard to
hydrolytic enzymes and methane production during
mesophilic anaerobic digestion of solid potato waste
(Parawira et  al., 2005). Some of the enzymes used by
microorganisms to degrade the potato were found to be
amylase, carboxymethyl cellulase, filter paper cellulase,
xylanase, pectinase, and protease. Both free and cell-
bound enzyme activities were measured. The activity of
the free enzyme was higher than that of the cell-bound
for all the enzymes. The amylase activity was highest,
followed by carboxymethyl cellulose, and filter paper
cellulase, while the other hydrolytic enzymes had low
activities. Confer and Logan (1998) concluded that the
complex insoluble substrate macromolecules such as
protein and polysaccharides are degraded by cell-bound
hydrolysis and followed by the release of hydrolytic
fragments into the bulk solution. This cell-associated
­hydrolysis and release is repeated until hydrolytic frag-
ments are small enough to be assimilated by cells.
Palmisano et  al. (1993) studied the distribution
and relative abundance of hydrolytic enzymes in
­several landfilled refuse sites. Esterases, proteases and
amylases were present in all of the samples. Enzyme
screening assays utilizing the API-ZYM test system
showed the incidence of enzymes in the order: specific
­phosphatases > esterases > gycosyl hydrolases. Analyses
of cellulose by the ­cellulose-azure test showed limited
­distribution of ­cellulases. Landfills are very heteroge-
neous ­environments; therefore it was not surprising to
find that enzymatic activities varied considerably among
BBTN 595384
refuse samples. The authors confirmed that that enzyme
activity may be a measure of the potential of landfill sites
to produce biogas as suggested by Jones et  al. (1983).
During two-stage anaerobic digestion of aerobic pre-
treated sisal leaf decortications residues, Mshandete et al.
(2008) reported that some of the enzymes produced by
microorganisms to hydrolyse the sisal leaf decortications
residues were pectinase, filter paper cellulase, amylase,
ß-galactosidase, caboxymethyl cellulase, xylanase, and
protease. Sisal leaf decortications residue is one of the
most abundant agro-industrial residues in Tanzania and
could be a potential source of feedstock to produce ­biogas.
Singh et  al. (1995) investigated the microbial load and
enzyme activities during production of biogas from night
soil at psychrophilic temperature. Among the hydrolytic
microbial population, proteolytic bacteria were the most
dominant (1 × 107/ml) followed by lipolytic (1.2 × 106/
ml), amylolytic (1.1 × 105/ml), and cellulolytic bacteria
(9 × 103/ml). The dominance of proteolytic bacteria was
expected due to the high protein content of night soil.
Amylase, CMCase, and lipase activities were some of the
hydrolytic enzymes analysed and at 10°C were about one
third of the activities of the same sample at 30°C. Night
soil is one of the most abundant particularly in highly
populated countries and is a potential source of biogas.
An understanding of the endogenous enzyme ­activities
in terms of their distribution and relative ­activity in
anaerobic digestion may result in improvement biogas
production from high-solid organic matter.
Several different types of enzymes have been reported
in sludge, such as aminopeptidases, galactosidases,
lipases, and phosphatases (Cadoret et al., 2002; Frolund
et al., 1996; Goel et al., 1998). The hydrolytic rates of these
extracellular enzymes have been reported as the overall
rate-limiting step for the mineralisation of organic ­matter
in the sludge treatment process (Whiteley et  al., 2002;
Vavilin et  al., 1996). The presence of enzymes such as
­glucosidase, cellulose, and protease break the bonds of
the extracellular polymeric substances in sludge result-
ing in deflocculation and improved anaerobic digestion.
Prospects, bottlenecks and perspectives
Enzyme hydrolysis is a mild, quick, and potentially
applicable method for complex biomass hydrolysis.
This method is specific and quite effective if the right
enzyme types, operating conditions, dosage, and
enzyme-waste ratios are optimal. Cell-free enzymes
offer several advantages over the use of microorgan-
isms in the treatment of waste biomass sludge. Enzymes
are capable of acting in the presence of various toxic
and recalcitrant substances and under a wide range
of environmental conditions, such pH, temperature,
and salinity (Gianfreda and Rao, 2004; Ruggaber and
Talley, 2006). Enzymes are able to act in a large range
of ­environmental conditions and remain active even
if these conditions quickly change (Ahuja et  al., 2004;
Gianfreda and Rao, 2004). Enzymes can work in the
Critical Reviews in Biotechnology
 Enzyme research and applications in biotechnological intensification of biogas production  11
presence of microorganisms, predators, and ­inhibitors
of microbial metabolism. Enzymes have easier access to
the substrate than microbes do, since they are smaller,
soluble, and more mobile in the sludge biomass
­environment. The benefits and disadvantages of using
enzymes in disintegrating biomass are summarised in
Table 2.
Other potential advantages of using enzymes include
absence of delays associated with the acclimatization
of biomass and the ease and simplicity of controlling
the process. Use of microorganisms would lead to
­biomass generation thereby creating another ­problem,
while enzymes reduce the volume of the waste (Ahuja
et  al., 2004; Karam and Nicell, 1997). Another very
important aspect of enzymatic treatment is that the
enzymes themselves are biodegradable proteins,
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
solutions. To overcome these problems, enzymes such
as lipases can be immobilised on a suitable media such
as ­alginate, which is cheap. Immobilized enzymes
have the ­advantages of multiple usage, controlled
­reactions, and thermostability (Jeganathan et  al.,
2007). In ­addition, for continuous operation in packed
bed ­reactors or fluidized bed reactors, immobilized
enzymes such as lipases yield a higher dosage per unit
volume of reactor and hence provide higher volumetric
production activity compared to free enzymes.
The addition of a wide range of commercial enzymes
to complex organic matter improves their ­hydrolysis
and solubilization resulting in improved biogas
­production. Enzymes act on specific substances ­present
in complex organic matter such as ­lignocellulosic
­matter, ­municipal, and industrial sludge therefore

meaning that enzymes that are not recovered after use
will degrade in the environment. The use of enzymes
is also ­desirable because they can perform the same
function as many harsher chemicals, but at neutral
pH, moderate temperature, and without production
of hazardous waste. However, enzymes tend to bind to
the solid waste matrix so that added enzymes might be
unevenly distributed in the solid waste environment.
Enzyme activity can also be lost through entrapment
by binding to the solid matrix, thermal denaturation,
and active site inactivation, loss of cofactors or pros-
For personal use only.
make them easily ­converted to biogas. However,
sludge, ­lignocellulosic, and food waste biomass are
a complex matrix of polymers and other substances
and efficient hydrolysis requires not only enzymes
but also ­optimization of the conditions and the opti-
mal enzyme mixture mostly tailor made and adjusted
to fit the kind of biomass being digested. Janin et  al
(1992) have shown that the contact between hydrolytic
enzymes and their substrates are crucial to anaerobic
digestion of ­complex ­particulate matter. The enzymes
would include ­cellulolytic enzymes, ­glycosidases,

thetic group, and reversible and irreversible inhibition
(Ahuja et  al., 2004; Aitken, 1993; Gianfreda and Rao,
2004). Although enzymes can survive in a wide range
of environments, they are not able to adapt themselves
to survive in environments that are outside their range
like microorganisms can. Free enzymes are generally
soluble and unstable, hence can be used once only in
­proteases, and lipases, etc. A lot of research still needs
to be carried out on the development and optimiza-
tion of ­enzymatic hydrolysis of complex biomass into
fermentable sugars. Much of the research has been
done at the laboratory-scale and there is little work
in pilot-scale and full-scale investigations on the use
of enzymes to hydrolyze ­biomass into fermentable

Table 2.  Summary of advantages and disadvantages of using enzymes to hydrolyse organic rich waste.
Advantages
Organic rich wastes hydrolysis using enzymes is done under mild, and non-corrosive and quick physical-chemical operating conditions
Enzymes are specific and effective unlike physical, mechanical and chemical methods
Process is compatible with other pretreatment options
Low energy requirement and low pollution, enzymes are biodegradable and non-toxic agents
Gives potentially high yields of simple sugar (75–85% of the theoretical maximum) and improvements are still projected (85–95%)
Cell-free enzymes can act in the presence of toxic and recalcitrant substances and under a wide range of conditions (pH, temperature)
Enzymes can work in the presence of inhibitors of microbial metabolism
Absence of acclimatisation period as for the microbial biomass
Easy to control the process
Sludge hydrolysis using enzymes leads to larger volume reduction since when microorganisms are used there is also biomass formation.
Enzymatic hydrolysis is not only energy sparing because of the relatively mild reaction conditions but also avoids the use of toxic and
corrosive chemicals
Low equipment maintenance costs
Disadvantages
The production of commercial enzymes is still costly, although the potential costs are being decreased.
Enzymatic pretreatment demands strict control of reaction conditions.
Enzyme activity is lost through binding to solid matrix, thermal denaturation, active site inactivation, loss of cofactors and reversible and
irreversible inhibition.
Some hydrolytic enzymes such as cellobiase are sensitive to inhibition by their own substrate and end product, cellubiose and glucose.
Biomass degradation is a highly complex multi-enzymatic process, there is still much to learn before enzyme cocktails with increased
activity can efficiently be developed

© 2011 Informa Healthcare USA, Inc. BBTN 595384

 12  Parawira Wilson
sugars for conversion to biogas. Pilot-scale and full-
scale research would ­facilitate better ­evaluation of the
technology, its constraints and ­opportunities. Different
studies in the literature are based on the use of different
types of biomass and a combination of these making
any ­comparison of the effectiveness of enzyme ­addition
difficult.
The cost of the enzymes is of prime importance in
order to realise the full potential of enzymatic treatment
of complex biomass. Reducing the cost and improving
the efficiency of separating and converting complex
materials into fermentable sugars is one of the keys to
a viable biogas production from such wastes (Merino
and Cherry, 2007). The enzymes that are presently being
investigated are still expensive because of the cost of their
production. However, this should not thwart the efforts
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by 41.197.25.6 on 08/19/11
to carry out more extensive research to identify the most
promising enzymes and determine the optimal condi-
tions for their application. In fact, the results of such
research should provide the incentive for commercial
development to finally produce the enzymes economi-
cally at large-scale. The costs are expected to decrease
as technology and techniques advance and as cheaper
growth substrates are explored for cultivating the parent
microorganisms.
The enzymes to be used in hydrolyzing complex wastes
such sludge and agricultural residues do not have to be
For personal use only.
highly purified as was demonstrated for lipid-rich wastes.
Such enzyme mixtures may include a variety of enzyme
activities capable of numerous catalytic ­functions for
them to be useful in heterogeneous substrates like sludge
biomass.
Conclusion
Anaerobic digestion provides biogas, a source of energy
to replace fossil fuels in many parts of the world using
local resources. However, utilization of complex mate-
rials such as agricultural crop residues, municipal solid
wastes, market wastes and animal wastes for biogas
production is limited by the hydrolysis step to convert
them into fermentable structures. This review has given
an update of the biotechnological advances towards
improved biogas production using enzymes. During
anaerobic degradation, acidogenic bacteria excrete
hydrolytic enzymes which enable the degradation
of complex organic matter into smaller compounds.
Thus, biological pretreatments using enzymes have
been studied with different materials to intensify bio-
gas production. Pretreatment of lignocellulosic agri-
cultural residues by cellulolytic and hemicellullolytic
enzymes have been reported to yield more biogas.
Similarly, there are several reports of improved biogas
production from lipid-rich waste after pretreatment
with lipases. The use of low-cost enzymatic prepara-
tions (lipases, cellulases, proteases) represents a vital
development in the treatment of waste and wastewater
with high organic matter, since the use of high-cost
BBTN 595384
commercial enzymatic preparations would make the
pre-treatment procedure economically infeasible.
There is currently considerable interest in developing
efficient and environmentally friendly ways of sludge
treatment from municipal treatment plants. Enzymatic
treatment of wastewater sludge biomass can improve
its solubilization, anaerobic digestibility, dewatering,
and hygienisation. Complex biomass hydrolysis is a
multi-enzymatic process which needs a combination
of the right enzymes, strategies of dosing, optimum pH
and temperature conditions. Future research needs to
focus on the development, optimization and validation
of the enzymatic hydrolysis of complex biomass at both
pilot-scale and full-scale. More opportunities exist for
improving enzyme mixtures, the stability, and activity
of the enzymes but also to reduce their cost and cost of
application.
Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
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