Anaerobic digestion is one of the most promising treatments for municipal sludge from the

standpoint of energy recovery. Meanwhile, it is also the main application at large- and medium-sized
WWTP currently.

Wastewater treatment plants and the energy production potential of sludge is greatly underutilized.
This is unfortunate since wastewater sludge production continuously provides an organic material
for anaerobic digestion that does not compete with other uses, as is otherwise the case with e.g.
energy crops. The produced sludge can be degraded and reduced through anaerobic digestion which
can be summarized to four main stages; hydrolysis, acidogenesis, acetogenesis and finally
methanogenesis. Extracellular enzymes, secreted by the microorganisms present in the anaerobic
digester are essential for hydrolysis of the particulate bioorganic molecules into monosaccharides,
amino acids and fatty acids. The substrate is thereby made accessible as nutrition for the
microorganisms in the anaerobic digester and is metabolized in several steps to the end products
methane and carbon dioxide

Anaerobic Digestion issues

Hydrolysis is recognized as rate-limiting step in the complex digestion process; therefore, pre-
treatment is necessary to make organic matter bioavailable to the microorganisms during digestion.

hydrolysis is considered to be the rate limiting step, resulting in the low degree of degradation
(Eastman and Ferguson, 1981). The low degradation is also partly due to the inherently short
hydraulic retention time (HRT) of the material in anaerobic digesters at WWTPs. Therefore, to
increase the degree of degradation within the available retention time, the hydrolysis rate of the
substrate has to be improved

Enzymatic solution

In AD systems, hydrolysis is considered the rate-limiting step, in which hydrolytic enzymes can attach
to the surface of the substrate and convert macromolecular organic compounds into small
molecules for further degradation. Therefore, enhancement of hydrolysis via pre-treatment or a
catalyst is crucial for improving methane production.

Enzymes produce biological processes which catalyse biochemical reactions that otherwise would
occur too slowly for keeping organisms alive. Enzymes can be selective and extremely efficient
catalysts that accelerate the rate of specific chemical reactions e such as nucleophilic, electrophilic,
and acid-base reactions by lowering the activation energy without being consumed. Thus, they do
not alter the equilibrium constant (final amount of product) but enhance the substrate's reactivity by
their functional groups. The protein nature of enzymes allows them to have a dual function as
catalysts and regulators of metabolic reactions.

Biological pre-treatment offers unique advantages compared to chemical or physical processes as it

is environmentally friendly and neither causes pollution nor needs special equipment. Biological pre-
treatment can be classified into 2 categories: (1) adding industrial (Yang et al., 2010) or endogenous
enzymes prior to anaerobic digestion processes; (2) adding specific bacteria which can secret certain
enzymes. It has been shown to result in improved degradation, dewatering properties of the sludge
and increase in methane production, and widely researched in lab- and in full-scale plants

As enzymes are essential for improving AcoD, supplying essential enzymes is an effective way to
enhance the methane yield. Studies have shown that the use of enzymes via both pre-treatment and
direct addition is feasible (Krishania et al., 2013; Parawira, 2012). Furthermore, enzymes can act in
the presence of various toxic, recalcitrant substrates, microorganisms, predators, and inhibitors of
microbial metabolism. Enzymes can remain active under a wide range of conditions (e.g., pH,
temperature, and salinity) even if these conditions change rapidly, thereby avoiding the adverse
effects that occur on living biomass. With smaller size, higher solubility and mobility, enzymes have
much easier access to substrates than microbes.

The deconstruction of lignin structures in the cell wall using microbes and/or enzymes as catalysts is
usually referred to as biological pre-treatment and occurs in the first stage of hydrolysis with other
pre-treatment processes. The use of cellulase enzymes for converting cellulose into oligomers and
sugar monomers is termed as enzymatic saccharification and occurs in the second stage of
hydrolysis. Keeping these biological processes separate is conceptually convenient, but it must be
considered that many of the relevant microbes simultaneously hydrolyse cellulose and lignin to
obtain carbon and energy from biomass. Effective biological pre-treatment requires various chemical
mediators and enzymes to address biochemical and physical barriers to hydrolysis; mixtures of
enzymes can work synergistically for expanding small pores and increasing access by opening the cell
wall matrix.

Previous studies have reported that adding enzymes into anaerobic digestion process could not only
cut down digesting time, improve sludge digestibility, and reduce disposal costs, but also could be
easily controlled, and its products were harmless to environment. Dey et al. (2006) demonstrated
that enzymes were useful both for releasing extracellular polymeric substances (EPS) and identifying
polysaccharides and glycoconjugates together with lectins panel. Hydrolytic enzymes can break
down polymeric substances through multi-step processes, during which compounds can be
transformed from a recalcitrant state to one that is more biodegradable. In other cases, enzymes are
able to increase the degradation rate of biodegradable substances, such as activated sludge,
allowing for more efficient treatment processes. Kim et al. (2006) investigated the effects of
enzymatic pre-treatment on solubilisation of food waste with commercial enzymes and obtained
good reduction efficiency. A number of researches had shown the benefit of additional enzymes on
the conditioning of wastewater solids and enhancement of the degree of dewaterability of
anaerobically digested biosolids.

To enhance hydrolytic processes and thereby increase the biogas production, with a concomitant
decrease of the amount of sludge for disposal, many chemical and physical, including thermal, pre-
treatment methods have been evaluated. However, since the dry content of untreated sludge
produced at WWTP is only between 0.5% and 3%, much of the required energy input, or addition of
chemicals, are therefore unproductive since it is to the largest part water that is being treated.

Enzymes can potentially be used without any additional energy input or changes to the conditions of
the substrate. However, that potential is only valid under the prerequisite that the enzymes added
have a high activity and a long enough life time at the conditions of use. The use of enzymes to
increase the accessibility of organic substances has been evaluated for a number of substrates, of
which some have been sludges from WWTPs. However, previous studies of enzymatic sludge pre-
treatment have shown diverse results, ranging from no improvements even in soluble chemical
oxygen demand (SCOD) to increased biogas production. Other studies have shown improvements on
the degradation of digester material upon addition of enzymes, however, at enzyme concentrations
that are not economically feasible.

Mixed-enzyme had great impact on sludge solubilisation than single enzyme. The mixture of two
enzymes (protease: amylase = 1:3) resulted in optimum hydrolysis efficiency, the efficiency of solids
hydrolysis increased from 10% (control test) to 68.43% at the temperature of 50 C.
Disadvantages of enzymes

The high-cost of commercial enzymatic preparation make the pre-treatment economically infeasible.
Thus, bioaugmentation (enhancement of the endogenous enzyme or enzyme-producing
microorganisms from the municipal sludge extracellular hydrolases) or inoculation of the extraneous
enzymeproducing stains is concerned as the proper way. While the effects of inoculation
thermophilic microorganisms on sludge solubilization and subsequent thermophilic anaerobic
digestion have been widely investigated, mesophilic pre-treatment by endogenous strain enzyme
has not been sufficiently researched. The vast majority of the anaerobic processes applied in practice
are still mesophilic.

Nevertheless, there is a surprisingly huge gap between the importance and the research efforts
made to elucidate the responsible enzymes for OMP biotransformation as was already stated by
Fischer and Majewsky (2014). This could be due to the extremely high complexity of biological
systems in STPs and to the lack of information about the involved enzymes. Actually, thousands of
enzymes exist, but less than 15% are described in detail, and only hundreds are commercially
available. To date, most studies have focused on activated sludge enzymes, particularly on
oxidoreductases. However, recent advances have been made on exploring the role of other
enzymatic activities such as hydrolases, enzymes acting on OMPs with amine groups, and enzymes
involved in bisphenol A degradation. Certain enzymes can be found in both aerobic and anaerobic
STP systems, such as hydrolases and transferases (e.g., hexokinase) whose activity do not depend on
redox conditions and are involved in several metabolic pathways; thus, some links between aerobic
and anaerobic biotransformation of OMPs could be found. Nevertheless, specific enzymes of each
process can be responsible for the reported biotransformation differences. Yet, to the best of our
knowledge, only one study (Gonzalez-Gil et al., 2017) so far investigated the OMP biotransformation
by enzymes present in microorganisms favoured under anaerobic conditions (namely by acetate
kinase).

The huge number of enzymes that could participate in a biological process, the interdependency of
their activities, and the complexity of OMPs, which usually have several functional groups
susceptible to be transformed, make it difficult to elucidate the enzymatic pathways involved in
OMP biotransformation. In this first approach, we found some links between the enzymatic activities
and the transformation of OMPs combining the knowledge about anaerobic enzymatic pathways
and OMP reactivity. However, further research focusing on identifying proposed TPs and performing
metaproteomic analysis is needed to corroborate these hypotheses, to identify more responsible
enzymes and to confirm the enzymatic reactions of OMPs in the basic lysates also occurring in
anaerobic digesters.(Gonzalez-gil et al. 2019)

Results

The majority of previous studies focused on the cellulase (cellobiohydrolase, endoglucanase and b-
glucosidase et al.), hemicellulase (endo-xylanase, acetyl xylan esterase and endomannanase et al.)
and ligninase (laccase, manganese peroxidase and lignin peroxidase et al.) (Schroyen et al., 2014,
2015; Van Dyk and Pletschke, 2012). In recent studies, cellulase pre-treatment increased the
methane yield of microalgae by 8%, resulting in a methane yield of 203.0 ± 0.4 mlCH4/gVS (Passos et
al., 2016); cellulase and xylanase pre-treatment increased the methane yield of the filamentous
algae by 17% and 4% respectively, resulting in a methane yield of 133 ± 4 mlCH4/gTS and 118 ± 5
mlCH4/gTS (Ehimen et al., 2013); the mix of laccase and versatile peroxidase pre-treatment
increased the methane yield of corn straw by 8%, resulting in a methane yield of 223.6 mlCH4/gVS
(Schroyen et al., 2015). Protease was mainly used in the treatment of algae for the cell wall
hydrolysis. Protease pre-treatment raised the methane yield of Chlorella vulgaris to 255.6 ± 4.9
mlCH4/gCOD, which was an increase of 51% (Mahdy et al., 2014). Amylase pre-treatment increased
the methane yield of the filamentous algae to 121 ± 9 mlCH4/gTS, which was an increase of 7%
(Ehimen et al., 2013).

Among the treatments, AcoD with added amylase (AMY) performed best with the highest methane
yield of 377.63 mlCH4/ gVS (see Fig. 1), which was an increase of 110.79% compared with CON1
(179.15 mlCH4/gVS). The second daily methane production peak achieved at the 5th day was the
highest. It was earlier than the control because of the improved hydrolysis of starch by amylase.

This study focused on the effect of enzymes on the methane yield of AcoD and its microbial
community. The direct addition of amylase and cellulase pre-treatment increased bio-methane
yields by 110.79% and 103.20%, respectively. With enzyme enhancement on AcoD, the hydrolytic
bacteria slightly changed, but the dominant microbial communities were similar. The results
demonstrated that all the three enzymes had no harmful effect on the microbial community.