HYDROLYSIS KINETICS IN ANAEROBIC DEGRADATION OF PARTICULATE

ORGANIC MATERIAL: AN OVERVIEW

V.A. Vavilin1, B. Fernandez2, J. Palatsi2, X. Flotats*2

1
Water Problems Institute of the Russian Academy of Sciences, Moscow 119991, Russian
Federation;
2
Laboratory of Environmental Engineering, Centre Udl-IRTA, University of Lleida, Avda Rovira Roure
191, E-25198 Lleida, Spain

Abstract

Applicability of the different kinetics to hydrolysis of particulate organic material in anaerobic

digestion is discussed. For complex substrate the first-order kinetics should be corrected taking

into account hardly degradable material. It was shown that the models describing hydrolysis

coupled to the hydrolytic bacteria growth works better at high or changeable organic loading.

The surface-related two-phase and the Contois models fitted well to experimental data on rice

grains, brewery-spent grains, starch and household solid wastes. Positive effect of particle

comminution with different shape of the particles (cylindrical and spherical) was demonstrated.

The examples of hydrolysis inhibition of municipal and slaughterhouse solid wastes were

shown. During complex waste degradation not hydrolysis but acetogenesis or methanogenesis

might be the rate-limiting stages. In such cases, a stimulation of hydrolysis (mechanically,

chemically or biologically) may have a negative effect causing an inhibition of these stages and

hydrolysis finally.

Keywords: anaerobic digestion, hydrolysis, kinetics, rate-limiting step, inhibition

*Corresponding author. E-mail address: xavier.flotats@irta.es

 1. Introduction

The intensive studies are performed now to improve efficiency of anaerobic digestion of solid

wastes (Mata-Alvarez, 2003; Hartmann, Ahring, 2005). Anaerobic degradation of complex

organic material has been described as a multi-step process that involves the stages of polymer

hydrolysis, acidogenesis, acetogenesis and methanogenesis (Batstone et al., 2002). For

description of hydrolysis the first-order kinetics of substrate degradation that reflects the

cumulative effects of many processes is the simplest type and has been traditionally used

(Eastman and Ferguson, 1981). However, in some papers it is underlined that hydrolysis still

remains the less defined step in the anaerobic digestion process (Miron et al., 2000; Gavala et

al., 2003). Maximum anaerobic degradability and fast rate of hydrolysis was reached in tests

with a high inoculum-to-substrate ratio (Fernandez et al., 2001).

In this paper, we showed that the models describing hydrolysis coupled to the hydrolytic

bacteria growth as well as taking into account substrate heterogeneity work better at high or

changeable organic loading than the traditional first-order kinetics. Inhibition of hydrolysis

should be taken into account if high concentration of particulate substrate is in the reactor.

2. Desintegration, solubilisation and enzymatic hydrolysis

In most of the cases presented in the literature a general term hydrolysis unites such terms as

disintegration, solubilisation and enzymatic hydrolysis (Batstone et al., 2002). Particulate

carbohydrates, proteins and lipids as well as the particulate and soluble inert material are the

products of disintegration of composite material. Monosaccarides, amino acids, long chain fatty

acids and glycerol are the products of the enzymatic degradation of particulate carbohydrates,

proteins and lipids, respectively, in which microorganisms benefit from the soluble products and

produce the corresponding hydrolytic enzymes.

2
 Hydrolysis of organic polymers is carried out by extra cellular enzymes (hydrlases). The

parallel enzymatic steps with cellulases, proteinases and lipases should be considered to account

for the difference in hydrolysis rate of the particulate carbohydrates, proteins and lipids,

respectively (Stryer, 1988). During degradation of monosaccarides, amino acids and long chain

fatty acids (hydrolysis products) volatile fatty acids (acetate, butyrate, propionate, lactate, etc.)

and hydrogen being precursors for methane production are formed.

The stage of acidogenesis that follows by hydrolysis is usually the quickest step of

anaerobic digestion of complex organic material. For efficient methane production it is

important to have a balance between the rates of different steps in anaerobic digestion of

complex organic material. When a process is composed of a sequence of reactions, one step

may be much slower than the other steps (Hill, 1977). The slowest (rate-limiting) step in

anaerobic digestion with suspended organic material is normally considered to be the hydrolysis

of solids (Pavlostathis and Giraldo-Gomez, 1991). According to Batstone et al. (2002),

hydrolysis can be represented by two conceptual models:

a) The organisms secrete enzymes to the bulk liquid where they adsorb onto a particle or

react with a soluble substrate (Jain et al., 1992).

b) The organisms attach to a particle, produce enzymes in the vicinity of the particle and

benefit from soluble products released by the enzymatic reaction (Vavilin et al., 1996).

The Michaelis-Menton kinetics might be applied for hydrolysis of soluble substrate:

(1)

where are the substrate and enzyme concentrations, is the maximum hydrolysis

rate, is the maximum hydrolysis rate constant, is the half-saturation rate coefficient.

According to Goel et al. (1998), soluble starch hydrolysis followed the model (1) where the

enzyme concentration was proportional to the sludge concentration. The dominant mechanism

of particulate substrate degradation apparently is of type (b).

3
 3. The first-order kinetics of carbohydrate, lipid and protein degradation

The following system of differential equations describing hydrolysis as the first-order reaction

not directly coupled to the bacterial growth is considered:

(2)

where S is the volatile solids (VS) concentration, P is the product concentration, k is the first-

order rate coefficient,  is the conversion coefficient of VS to product. After integration the

product concentration is expressed as

(3)

where and are the initial product and substrate concentrations. A non-linear regression

may be used to estimate the values of coefficients k and  and their standard deviations.

Figure 1 shows the first-order kinetics of hydrolysis/acidogenesis for complex substrate

(cattle manure) at the thermophilic conditions (55oC) and at the different initial waste

concentrations. Acetate was the most significant product with concentration of ten times higher

than the other VFA. The data corresponded to the highest initial waste concentration (22%) was

used for calibration and other (16, 10 and 5%) were used for validation of the first-order

kinetics. We can say that the first-order kinetics fitted the data reasonably well.

During protein degradation ammonium nitrogen is released. Figure 2 shows the first-

order kinetics for gelatine at the thermophilic conditions (55oC) estimated from ammonia

nitrogen released during amino acids fermentation. In this case, rather high experimental errors

were obtained because of a small sample volume used for analysis. The system (2) with

methane as the final product can be used if hydrolysis becomes the slowest step in comparison

4
to the other steps of solids conversion to methane (acidogenesis, acetogenesis and

methanogenesis). In Figure 3, the methane volume released from complex waste was used to

estimate the values of hydrolysis coefficients.

Table 1 summarizes the typical values of rate coefficients taken from different literature

sources for different substrates with a wide range of the values of first-order rate coefficient for

composite and simpler organic materials including carbohydrates, lipids and proteins.

According to the Table 1, the rate coefficients for carbohydrates, proteins and lipid hydrolysis

differ very much. In the famous ADM1 model (Batstone et al., 2002) the disintegration rate of

composite material are around , but the selected values of carbohydrate, protein and lipid

hydrolysis are set up as the same value of in mesophylic conditions. The ADM1 model

was oriented mostly for description of anaerobic digestion of activated sludge. In such case the

influence of hydrolysis step in the model is completely excluded (Feng et al., 2005; Rusdi et al.,

2005). However, in analysis of particular substrates the different authors reported significantly

smaller values of the first-order hydrolysis rate coefficients of carbohydrates, proteins and

lipids.

Different factors affected on the rate coefficient values including the temperature.

Veeken and Hamelers (1999) showed that the average activation energy in the Arrhenius

equation for five biowaste components of that confirmed that the hydrolysis

rate of biowaste components is controlled by enzyme kinetics if the hydrolytic enzyme

concentration exceeds the concentration of degradable surface sites of the substrate.

4. Biodegradability of complex substrate

The first-order kinetics can only be applied when the rate-limiting factor is the surface of the

substrate, and bioavailability or biodegradability does not alter (Sanders et al., 2003). The

biodegradability and hence the biogas potential of the complex substrate depends on the content

5
of carbohydrates (including cellulose, hemicellulose and lignin fractions), proteins and lipids

(Angelidaki, Sanders, 2004). Cellulose is a main polymer in lignocellulosic biomass and in

many organic wastes. The rate of cellulose degradation depends on the enzymatic activity, as

well as on the physical-chemical conditions of the cellulose polymers (Klesov and Rabinovich,

1978). The biodegradability of particulate substrate is depended on the lignin content (Chandler

et al., 1980) and the structure of the lignocellulosic complex (Tong et al., 1990). It decreases

with the increase of the lingo-cellulosic content of the waste (Buffiere et al., 2005). South et al.

(1995) developed an enzyme-adsorption based kinetic model to describe saccharification and

fermentation of insoluble lignocellulosic substrates to ethanol. The model showed that the rate

of hydrolysis increases for increasing enzyme concentration and for increasing available

adsorption places. Kadam et al. (2004) described cellulase adsorption onto pretreated

lignocellulose via a Langmuir equation.

The ultimate biodegradability of substrate can be determined by long-term batch

digestion studies (Kayhanian, 1995) but the reaction period should be specified. Figure 4

showed applicability of the first-order kinetics corrected by non-degradable fraction for sludge

as the complex substrate:

(4)

where is the initial substrate concentration, is the non-degradable fraction of substrate.

Kayhanian et al. (1991) and Kayhanian and Tcshobanoglou (1992) reported that the

biodegradable fraction of volatile solids (VS) was 82% (food waste), 72% (yard waste), 82%

(office paper), 67% (mixed paper), 22% (newsprint). Two types of MSW (easy degradable and

recalcitrant) should be considered to model MSW degradation (Vavilin et al., 2006). The

famous model Activated Sludge Model no.2 considering enzymatic hydrolysis under anaerobic,

anoxic and aerobic conditions as the first step in complete metabolism of soluble and particulate

6
substances divides these substances on inert, rapidly and slowly hydrolysable (Henze et al.,

1995).

Instead of the model (4), the different types of kinetics including n-order reaction may be

applied to describe complex substrate hydrolysis. As an example, Figure 4 showed applicability

of the following equation:

(5)

where is the maximum hydrolysis rate which in turn depends on hydrolytic biomass or

enzymes concentrations, is the additional model coefficient.

The equation (4) can be corrected introducing biomass concentration :

(6)

where is the rate constant and is the power index. Figure 5 showed peptone degradation at

the different initial sludge concentration.

One important finding is that the substrate hydrolysis rate depends strongly on the origin

and the previous acclimation of the anaerobic culture (Gavala et al., 1999). The biochemical

pathways of different organic materials may be mutually affected. According to Breure et al.

(1986), a complete degradation of protein in the presence of carbohydrates often cannot be

achieved in anaerobic wastewater treatment.

5. The surface-related kinetics and two-phase model of hydrolysis of particulate substrate

Firstly, Hills and Nakano (1984) showed a linear relationship between the gas production rate

and inverse particle diameter for tomato solid waste with average particle diameters of 0.13 to

2.0 cm. Similar results were obtained by Shrama et al. (1988) with agricultural and forest

7
residues. Sanders (2001) confirmed that relationship for starch particles with much less

characteristic sizes. A decrease of particle radius R was described as a linear function with time.

(7)

where is the initial average radius, is the substrate density, is the surface based hydrolysis

constant, t is the time. However, using the function (7), the negative R values may be obtained at

a high time interval. Valentini et al. (1997) used the exponential relationship between rate

constant of cellulose hydrolysis and particle diameter:

(8)

where d is the particle diameter, , are the constants.

Vavilin et al. (1996) developed the following rate function of hydrolysis for different

shape particles:

(9)

where , are the current and initial substrate concentrations, n is the degree index that equals

to 2/3, 1/2 and 0 for spherical, cylinder and plate-form particles. The last case is equivalent to

zero-order kinetics. The hydrolytic constant is a function of the ratio between the characteristic

sizes of bacteria and particles hydrolyzed:

(spherical particles) (10)

(cylinder particles) (11)

where is the maximum specific hydrolysis rate; and are the bacterial and particle

densities, respectively; denotes the depth of the bacterial layer and is the current diameter

of particles. Thus, for different particle shape, a rate constant value is reciprocal to the

characteristic size of the particle.

8
 The complete enzymatic hydrolysis stage is a complex multi-step process for

carbohydrates, proteins and lipids, which may include multiple enzyme production, diffusion,

adsorption, reaction and enzyme deactivation steps. In the simplest first-order kinetics a rate of

hydrolysis does not depend on hydrolytic biomass concentration. So, that kinetics could not

describe a typical sigmoid curve when the hydrolysis rate increases in time. However, during

hydrolysis the particulate substrates make contact with hydrolytic microbial cells and the

released enzymes. Two main phases might be taken into account for a description of the

hydrolysis kinetics. The fist phase is a bacterial colonization, during which the hydrolytic

bacteria cover the surface of solids. Bacteria on or near the particle surface release enzymes and

produce the monomers that are utilized by the hydrolytic bacteria. The daughter cells fall off

into the liquid phase and then they try to attach to some new place on a particle surface. Thus, a

direct enzymatic reaction as the intermediate step of the total two-phase process may be rather

quick in comparison with the stages of bacterial colonization and surface degradation. When an

available surface is covered with bacteria the surface will be degraded at a constant depth per

unit of time (second phase).

The surface-related hydrolysis kinetics model that takes into account colonization of the

waste particles by hydrolytic bacteria has been developed by Vavilin et al. (1996).

(12)

where S is the volatile solid waste concentration, X is the concentration of hydrolytic

(acidogenic) biomass, is the maximum hydrolysis rate;  is the equilibrium constant equal to

the ratio between the adsorption and desorption rate constants in the Langmuir function; is

the half saturation coefficient for the volatile solid waste concentration S.

Microorganisms attached to a particle produce enzymes in the vicinity of this particle

benefiting from soluble products released by the enzymatic reaction. The Contois model that

9
uses a single parameter to represent saturation of both substrate and biomass is as good at fitting

the data as the two-phase model

(13)

where is the maximum specific hydrolysis rate; is the half saturation coefficient for the

ratio S/X. The surface-related (12) and Contois (13) models have the same limiting cases:

(i) exponential biomass growth (surface-related model:  X<<1, S >> ; Contois model:

S/X>> )

(14)

and (ii) first-order kinetics (surface-related model:  X>>1, S<< ; Contois model: S/X<< )

(15)

Introducing the hydrolytic biomass concentration the following system of differential equations

describing hydrolysis coupled to the bacterial growth is written instead of the model (2):

(16)

where X is the biomass concentration, Y is the biomass yield coefficient, is the rate

function depended on substrate and biomass concentration. In the system (16) a biomass decay

process was neglected. In such case, the system (15) can be integrated and a non-linear

regression gives the coefficient values and their standard deviations (Lokshina, Vavilin, 1999;

Vavilin et al., 2004; Lehtomaki et al., 2005). Figures 6-7 show an applicability of the two-phase

10
and the Contois equations for methane production data during starch and brewery-spent grains

degradation.

Palatsi et al. (submitted) described the hydrolysis/acidogenesis of proteins and lipids in

synthetic waste by the first order and the Contois kinetics, respectively. According to the model,

in two peaks of dynamics of acetate concentration measured in the experiments the second peak

occurred because hydrolysis rate of lipids increased in time due to biomass growth of

hydrolytic/acidogenic bacteria.

6. Mechanical pretreatment

To enhance anaerobic biodegradation several processes from comminution to cell desintegration

were used (Delgenes et al., 2003). Size reduction of the particles to increase the available

specific surface represents an option for accelerating the digestion process. Kayhanian and

Hardy (1994) indicated that the methane production rate was inversely proportional to the

feedstock particle size. Wen et al. (2004) showed that decreasing the particle size from 840-590

to 590-350 enhanced glucose yield by 29% after 96h-treatment of animal manure. However,

further decrease in particle size has no effect. Mechanical size reduction was found to be

efficient for enhancing the biogas potential from fibre-rich materials like maple leaves and hey

stems which ere difficult to digest (Palmowski and Muller, 2000) or wastes like manure

(Hartmann et al., 1999). However, Masse et al. (2003) showed that there was no significant

particle size effect on pork fat hydrolysis.

Fig. 8(a,b) shows a comminution effect on anaerobic digestion of hay using the first-

order and half-order kinetics of hydrolysis. The traditional first-order kinetics showed a better fit

to experimental data. In spite of significant reduction of characteristic size of particles (from

higher than 1.6 mm to less than 0.2 mm) the first-order and half-order kinetic constants increase

in 1.5 and 1.2 times, respectively. It is corresponded to an increase in ratio of surface areas of

11
1.3 (Palmowski et al., 2001). Evidently, that the cylinder particles are more appropriate in that

case. The specific methane production increases because of comminution. Bjornsson et al.

(2005) showed that pre-treatment of fibre waste fraction through size reduction down to 2 mm

increased biogas yield from to .

Fig. 9(a,b) shows a comminution effect on anaerobic digestion of rice grains using the

Contois and two-phase kinetics of hydrolysis. They showed about similar fit to experimental

data. Significantly higher initial hydrolytic biomass concentration (from 0.45 to 2.5 g/l for the

Contois kinetics and from 0.9 to 6.0 g/l for the two-phase kinetics) as well as the half-saturation

coefficients ( and ) was appointed to the case with comminution. It means that both

models transformed into the first-order model of hydrolysis. In that case an increase in ratio of

surface areas reaches to 2.9 (Palmowski et al., 2001). The specific methane production increases

because of comminution. In that case, probably the spherical particles are more appropriate for

the rice grains.

The other way to promote hydrolysis is the biological and physico-chemical pre-

treatments of substrate breaking the polymer chains into soluble components (Mace et al., 2001;

Delgenes et al., 2003; Park et al., 2005). In actual digester conditions, one does not know the

proportions of the particles with different shapes and the particles can be degraded from inner

and outer surfaces (Hobson, Wheatley, 1992). Thus, we may assume that the first-order model

of hydrolysis is the simple approximation of actual processes. The other models may be more

appropriate to experimental data. For example, Scober et al. (1999) observed zero-order kinetics

of hydrolysis in the acidogenic reactor during the two-stage anaerobic digestion of municipal

solid wastes.

7. Steady-state models

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 Assuming the first-order kinetics of hydrolysis as the rate-limiting step in anaerobic

digestion the following equations are used for substrate (suspended solids) and specific methane

production for a complete-mixing stirred-tank reactor (CSTR) operating at steady state:

(16)

(17)

where , are the influent and effluent solids concentration, B is the specific methane

production and T is the solids retention time. The conversion coefficient  of VS to product P

for the ultimate specific methane production is written as . Ristow et al. (2004) showed that at

changeable influent primary sludge concentration a standard deviation of the first-order kinetic

coefficient was relatively large . Eastman and Fergusson (1981) were the

first who used the equation (16) for estimation of particulate sludge reduction measured in COD

units introducing additionally the refractory coefficient for non-degradable fraction of

particulate substrate.

Chen and Hashimoto (1978) developed the following equations for the solid substrate

concentration and specific methane production for a CSTR digester operating at steady state:

(18)

(19)

where is the model coefficient, Y is the biomass yield coefficient, is the maximum specific

substrate removal rate and is the maximum specific growth rate of biomass. In fact, the

equations (18) and (19) based on the Contois kinetics (13) taking into account that a biomass

value is depended on difference between the initial and current substrate concentrations

and the coefficient K is presented as . The applicability of the Contois

13
kinetics for the description of anaerobic digestion of particulate organic matter was shown in a

number of papers (e.g Domenech, Flotats, 1997; Vavilin et al., 2001).

The Chen and Hashimoto as well as the Contois models describe a washing out

phenomenon of the hydrolytic biomass in CSTR reactor if

(20)

Introducing the refractory coefficient R for non-degradable fraction of particulate substrate into

(20) Chen and Hashimoto (1978) obtained the following equation:

(21)

Number of authors used the Chen and Hashimoto model (Hill, 1982; Samson, Leduy, 1986;

Lema et al., 1987; Maraval, Vermande, 1990; Flotats, 1993). However, it was found (Hashimoto

et al., 1979) that the waste type, temperature and influent solids concentration affected on the

values of , and K.

At the Chen and Hashimoto model transforms into the first-order model with the

rate coefficient .

8. Inhibition of hydrolysis

The models (2, 16) were written assuming that hydrolysis was the rate-limiting stage in

anaerobic digestion and the stages of acidogenesis, acetogenesis and methanogenesis were not

rate-limiting. The case with hydrolysis as the rate-limiting step described by the Contois kinetics

for waste and residual organic material in inoculum is presented in the Fig. 10. Reasonably good

modeling results were obtained using for calibration only two sets of experimental data of 28.8

g/l VS and inoculum alone.

During solids degradation instead of hydrolysis the stages of acetogenesis and

methanogenesis may be the rate-limiting. In such cases a rate of the product formation P is not

14
strictly depended on the hydrolysis rate. A sigmoid type of methane accumulation curves

presented in Fig. 10 can be simulated if the acetoclastic methanogenesis is assumed to be the

rate-limiting step described by the Monod model (Fig. 11). However, such assumption was not

valid for all data set with different initial waste concentrations (compare Figs. 10 and 11).

During anaerobic digestion of pork fat in slaughterhouse wastewater LCFA oxidation

was the rate-limiting step (Masse et al., 2002). Thus, mechanical or enzymatic hydrolysis pre-

treatments of fat-containing wastewaters should not substantially accelerate anaerobic treatment.

Vavilin and Angelidaki (2005) demonstrated that during household solid waste degradation

initially acetoclastic methanogenesis was the rate-limiting stage. To explain the negative effect

of vigorous mixing they hypothesized that spatial separation of the initiation methanogenic

centers from active methanogenic zones is the key factor for efficient anaerobic decomposition

of solids at high organic loading rates. If methanogenesis is the rate-limiting step during the

start-up period, it is better to avoid vigorous mixing that suppresses growth and propagation of

methanogenic centers over the reactor volume. In such case, an increase of the initial hydrolysis

rate above a critical value cases an inhibition, first of methanogenesis and then of hydrolysis

(Vavilin et al., 2003). A decrease of the initial methanogenesis rate below a critical value has the

same effect. Thus, stimulation of hydrolysis may have a negative effect on solids conversion to

methane. If hydrolysis becomes the rate-limiting step, a high mixing may enhance methane

production and solids degradation.

Different types of inhibition by high values of VFA, LCFA, H2, NH3 as well as acidic

and alkaline pH were observed in anaerobic digesters (Batstone et al., 2002; Lokshina et al.,

2003) mostly for acetoclastic methanogens and acetogens. Much less attention was paid to

inhibition of hydrolysis. Hydrolysis can be inhibited by the accumulation of amino acids and

sugars (Sanders, 2001; Kadam, 2004). During cellulose degradation cellobiose as the

intermediate product may be stronger inhibitor than glucose (Duff, Murray, 1996). Other

15
possible inhibitors also are non-ionized VFA (De Baere et al., 1985; Brumeler et al., 1991).

Llabres-Luengo and Mata-Alvarez (1988) showed that high concentration of VFA accompanied

by low pH might inhibit hydrolysis. Veeken et al. (2000) reported that actual inhibitor is the

acidic pH, but not VFA or non-ionized VFA, however, a scatter of data used for analysis was

rather high. They proposed a linear function of pH inhibition in the interval between 5.0 and 7.0

(22)

where is the first-order coefficient in day -1. However, such function could not describe a full

stop of the process at the pH about 5.6. For that purpose a generalization of the non-competitive

inhibition function introduced into the rate coefficient is more suitable (Vavilin and Angelidaki,

2005)

(23)

where I is the inhibitor concentration, KI is the inhibition constant, n is a degree index.

While Breure et al. (1986) and Yu and Fang (2003) concluded that VFA do not inhibit

protein degradation, using gelatine as substrate, Gonzalez et al. (2005) clearly show that acetic

acid reduced the gelatine hydrolysis rate in mesophilic, saline environment. Describing an

inhibition of hydrolysis they showed that the n-order Levenspiel and Luong models showed

much better fit to experimental data than the traditional non-competitive model. Controversially,

Flotats et al. (2005) showed that no inhibition by VFA occurred during protein hydrolysis. The

low pH and high lipid concentration could also affect the hydrolysis (Palenzuela-Rollon, 1999).

It has been stated that lipid hydrolysis hardly occurs without methanogenic bacteria that keep

non acidic pH and low VFA value. Lu et al. (2004) studied enzymatic activity during start-up of

dry anaerobic mesophilic and thermophilic digestions of organic fraction of municipal solid

waste. It was shown that the lower hydrolysing protease activity during the first 2-3 weeks was

16
due to the inhibition of the low pH, but was enhanced simultaneously later with the pH increase.

Thermophylic digestion recovered quicker from temporary acidic pH.

Jonsson et al. (2006) modelling MSW decomposition in the 100-L landfill simulation

reactors concluded that inhibition of the hydrolytic and methanogenic processes occurred during

the acidogenic phase. The degradation of the readily degradable waste fraction generated

conditions that inhibited further degradation of the recalcitrant waste related to low pH of 5.6.

Addition of methanogenic inoculum removed a few years delay in solids decomposition

decreasing VFA concentration. At the same time phthalate esters biodegradation started. The

hydrolysis of phthalate diesters requires to be described coupled with hydrolytic biomass

growth. In that case, according to Vavilin et al. (2005), the coefficient for biomass yield Y

corresponds to the intermediate product (alcohol). Much worse fit of the model to phthalate

monoester data was obtained using the traditional first-order kinetics of diester and monoester

hydrolysis (Vavilin et al., 2006; Jonsson et al., 2006). In the parallel reactor the acidogenic

phase was avoided by preliminary aeration reducing VFA level and increasing pH.

Lokshina et al. (2003) showed a temporary inhibition of solids hydrolysis during

household (HSW) and slaughterhouse solid (SSW) wastes degradation. An inhibition of

hydrolysis happened at acidic for HSW with 70.6 % of carbohydrates, 19% of proteins and

9.9% of lipids, and at neutral and alkaline pH for SSW with 50% of proteins, 31.4% of lipids

and 18.6% of carbohydrates. By simulations it was shown that most likely that hydrolysis was

inhibited by high VFA concentration in the case with HSW and by high LCFA concentration in

the case with SSW that increase during hydrolysis and acidogenesis of easily degradable waste.

Both systems recovered when biomass growth of methanogens occurred.

The influence of different ratios of lipids (from 5 to 47%) on hydrolysis of artificial

waste was studied by Cirne et al. (2004). An inhibition was observed at the high amount of

lipids (31% and higher). Studying the affect of lipase addition on enzymatic hydrolysis of lipids

17
it was shown that the higher the enzyme concentration, the more accelerated was the inhibition

of the methane production. The enzyme addition enhances the hydrolysis but the produced

intermediates (LCFA) are causing inhibition of the later steps of anaerobic digestion and finally

the first step, i.e. hydrolysis. This work demonstrated that a stimulation of hydrolysis may have

a negative effect if all stages are not in balance. A negative correlation between the hydrolysis

rate and the methane production was observed by Neves et al. (2005). The products of the

alkaline hydrolysis were less toxic and/or inhibitory for the subsequent stages of anaerobic

digestion process.

In the two-stage (often also called two-phase) digestion, the acidogenic stage is spatially

separated from the methanogenic stage by using two consecutive reactors (Ghosh and Pohland,

1974). Because the growth rate of acidogens is much higher than that of methane-formers a

kinetic control is used in order to accomplish stage separation when substrate is dissolved or

readily hydrolysable. However, if a complex organic substrate is used in the feed, the

advantages of a two-stage system are not so evident (Hanaki et al., 1987). Vavilin et al. (2001)

reported that Contois kinetics is preferable to the traditional first-order kinetics when

considering the optimal design of a two-stage anaerobic digestion system. In the acidodenic

stage the solids hydrolysis may be inhibited, thus, a recycle from methanogenic stage will be a

good solution to avoid such inhibition (e.g. Wang et al., 2004). In the two-stage anaerobic

digestion process, the process should be carried out when the undissociated acid concentration is

between the inhibitory level of acidogenesis and methanogenesis (Babel et al., 2004). Using

additional first stage of aerobic solubilization in the 3-stage fermentation system treated food

waste, better efficiency in methane production was obtained (Sakamoto et al, 2004) probably

because of excluding potential inhibition by high VFA level arisen during acidogenic stage.

At present, for anaerobic digestion of solid waste, is still not possible to single out

specific processes as all-around and optimally suited under all circumstances because of the

18
complexity of the biochemical pathways involved and novelty of technology (Vandeviere et al.,

2003; De Baere, 2005).

9. Conclusions

The first-order kinetics is traditionally used but it may be invalid to describe solids hydrolysis.

For complex substrate the first-order kinetics should be corrected taking into account hardly

degradable material. The Contois kinetics considering a growth of hydrolytic/acidogenic

biomass as well as two-phase kinetics taking into account the phases of surface colonization and

degradation showed a better fit to experimental data at high or changeable organic loading rate.

At high organic loading an inhibition of hydrolysis should be considered when analysis of

efficiency of anaerobic digestion of complex substrates is carried out. In such case,

methanogenesis or acetogenesis can be the rate-limiting steps in anaerobic digestion. To

describe it a more complex structured model should be used.

Acknowledgements

This research was supported by the Spanish Ministerio de Educación y Ciencia (Project REN

2004-00724).

19
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 Figure Captions

Fig. 1. Time profiles of the acetate concentration during cattle manure degradation at the

different initial waste concentrations (in % of weight units). Symbols refer to the experimental

data and lines to the model predictions at k = 0.13 d-1,  = 0.18 g acetate g-1 VS. Data were taken

from Montane (2001).

Fig. 2. Time profile of the ammonia concentration during hydrolysis of gelatine. Symbols refer

to the experimental data and lines to the model predictions at k = 0.7 d-1,  = 0.14 g N-NH4 g-1

VS. Initial concentration of gelatine was 5.7 g COD/l. Data were taken from Flotats et al.

(2005).

Fig. 3. Time profile of the methane volume released during pig slurry (80%) and pear waste

(20%) degradation at the thermophilic conditions (55oC). Symbols refer to the experimental data

and lines to the model predictions at and . Data were

taken from Campos et al. (2001).

Fig.4. Time profile of the carbohydrate concentration during disintegration and hydrolysis of

sewage sludge. Symbols refer to the experimental data and lines to the model predictions at

, (model (4), solid line) and , (model (5), dashed

line). Data were taken from Park et al. (2005).

Fig.5. Time profile of the peptone concentration at the several initial relative sludge

concentrations. Symbols refer to the experimental data and lines to the model (6) predictions at

, . Data were taken from Sanders (2001).

31
Fig.6. Time profiles of the efficiency of starch hydrolysis. Symbols refer to the experimental

data and lines to the two-phase model model predictions assuming the first-order kinetics of

surface degradation at and ,  = 20 l g-1 VS, . Data

were taken from Sanders (2001).

Fig. 7. Time profiles of the methane volume released during degradation of brewery-spent

grains at the mesophilic conditions (37oC). Symbols refer to the experimental data and lines to

the model (16) predictions with the Contois kinetics at , ,  = 115 ml

CH4 g-1 VS, . It is assumed that 100 ml of CH4 formed quickly from soluble

organic material presented initially. Data were taken from Fernandez (2001).

Fig. 8. Time profiles of the specific methane volume released during degradation of hay at the

mesophilic conditions (35oC) with and without comminution. Symbols refer to the experimental

data (Palmowski et al., 2001) and lines to the model predictions:

first-order kinetics without comminution (k = 0.1 d -1, = 550 ml g-1 VS),

first-order kinetics with comminution (k = 0.15 d -1,  = 590 ml g-1 VS),

half-order kinetics without comminution (k = 0.075 d -1,  = 530 ml g-1 VS),

half-order kinetics with comminution (k = 0.090 d -1,  = 600 ml g-1 VS).

Fig.9. Time profiles of the specific methane volume released during degradation of rice grains at

the mesophilic conditions (35oC) with and without comminution. Symbols refer to the

experimental data (Palmowski et al., 2001) and lines to the model predictions:

32
Contois kinetics (without comminution: , ,  = 620 ml CH4 g-1 VS,

; with comminution: , ,  = 670 ml CH4 g-1 VS,

),

two-phase kinetics (without comminution: , , ,

 = 600 ml CH4 g-1 VS, ; with comminution: ,

, , = 660 ml CH4 g-1 VS, ).

Fig. 10. Methane accumulation curves during household solid waste anaerobic digestion in

batch reactors with 0 (asterisk), 4.8 (point), 9.6 (cross), 14.4 (six-point star), 19.2 (plus sign), 24

(square) and 28.8 (circle) in g/l for VS. Symbols: experimental data (Angelidaki et al., 2005);

curves: batch model prediction with the Contois kinetics of hydrolysis according to Lokshina et

al. (2005): waste: ; residual organic material in inoculum:

, .

Fig. 11. Methane accumulation curves during household solid waste anaerobic digestion in

batch reactors with 0 (asterisk) and 28.8 g/l VS (circle). Symbols: experimental data

(Angelidaki et al., 2005); curves: batch model prediction with the first-order kinetics of

hydrolysis (waste: ; residual organic material in inoculum: ) and Monod

kinetics for acetoclastic methanogenesis ( , , ,

).

33
 Table 1. Kinetic coefficients of the first-order rate of hydrolysis.

Substrate T, oC References
Carbohydrates 0.025-0.2 55 Christ et al., 2000
Proteins 0.015-0.075 55   
Lipids 0.005-0.010 55   
Carbohydrates 0.5-2.0 Garcia-Heras, 2003
Lipids 0.1-0.7   
Proteins 0.25-0.8   
Lipids 0.76 Shimizu et al., 1993
Lipids 0.63 25 Masse et al., 2002
Cellulose 0.04-0.13 Gujer, Zehnder, 1983
Cellulose 0.066 35 Liebetrau et al., 2004
Kitchen waste 0.34 35   
Biowaste 0.12 35   
Cattle manure 0.13 55 Present study
Pig manure 0.1 28 Vavilin et al., 1997
Gelatine 0.6 55 Flotats et al., 2005
Municipal solid waste 0.1 15 Bolzonella, 2004
Office paper 0.036 35 Vavilin et al., 2004
Cardboard 0.046 35   
Newsprints 0.057 35   
Food waste 0.55 37   
Forest soil 0.54 30 Lokshina, Vavilin, 1999
   0.09-0.31 20   
Slaughterhouse waste 0.35 35 Lokshina et al., 2003
Household solid waste 0.1 37 Vavilin, Angelidaki, 2005
Primary sludge 0.4-1.2 35 O´Rourke, 1968
   0.99 35 Ristow et al., 2004
Secondary sludge 0.17-0.60 35 Ghosh (1981): referenced by
Gavala et al., 2003
Crops and crop residues 0.009-0.94 35 Lehtomaki et al., 2005

34
Fig. 1.

35
Fig. 2.

36
Fig. 3.

37
Fig. 4

38
Fig. 5

39
Fig. 6.

40
Fig. 7.

41
Fig. 8.

42
Fig. 9.

43
Fig. 10

44
Fig. 11

45