A MASS DIFFUSION-BASED INTERPRETATION OF THE EFFECT OF

TOTAL SOLIDS CONTENT ON SOLID-STATE ANAEROBIC DIGESTION

OF CELLULOSIC BIOMASS
F. Xu

The author is Fuqing Xu, ASABE Member, Graduate Student, Department of Food, Agricultural and Biological Engineering, The Ohio State

University, OARDC, Wooster, Ohio, USA. phone: 330-234-8006; e-mail: xu.467@ osu.edu.

ABSTRACT.

Compared to traditional liquid anaerobic digestion (AD), solid-state anaerobic digestion (SS-AD) can reduce reactor

volume as well as the water and energy consumption, and thus potentially increasing the economic benefits of AD.

However, to-date, very few modeling system has been developed for describing SS-AD. In this study, the volumetric

methane production rate in SS-AD of cellulosic biomass was found to have a threshold at the total solids (TS) content

between 15-20%, which cannot be explained by the conventional liquid AD theory. This study hypothesized that the high

TS contents beyond the threshold retarded mass diffusion and resulted in the accumulation of hydrolysis products, e.g.

sugar, which in turn inhibited hydrolysis and reduced the overall methane production rate. Based on this hypothesis, a new

SS-AD model was developed to take into account the mass diffusion limitation and hydrolysis inhibition. The results

predicted by the model showed a good agreement with the experimental data and literature-based data, verifying that the

observed reduction of methane production in SS-AD could be ascribed to hydrolysis inhibition as a result of the mass

diffusion limitation.

Keywords. Hydrolysis, inhibition, diffusion, biomass, anaerobic digestion, methane.

INTRODUCTION

Anaerobic digestion (AD) is a biological process that converts organic matter into methane-rich biogas as a renewable

energy source (Motte et al., 2013). Based on the total solids (TS) content used in digester operation, AD can be

categorized into liquid AD (L-AD) and solid-state AD (SS-AD) (Li et al., 2011). L-AD operates at TS ranging from 0.5%

to 15%, and is a traditional technology that has been widely used to treat sewage sludge, food waste, and animal manure;

while SS-AD operates at TS greater than 15%. SS-AD has gained more attention in recent years due to the increasing

1
market demand for treating solid organic wastes, such as the organic fraction of municipal solid waste and crop residues

(Karthikeyan and Visvanathan, 2013). SS-AD is advantageous over L-AD in solid waste handling as it allows much higher

loading in a smaller volume with less energy input and water addition (Abbassi-Guendouz et al., 2013). Moreover, the

compost-like digestate remaining after SS-AD is easier and less costly to transport (Li and Wang, 2011). The fibrous

biomass that may cause floating and stratification problems in L-AD can also be easily handled by SS-AD (Forster-

Carneiro et al., 2007). Due to these advantages and its simple structure, SS-AD has been widely used in Europe,

representing about 60% of the total AD treatment capacity in 2010 (De Baere et al., 2010).

While the application of SS-AD holds promise for solid waste treatment and economical bioenergy production, it has

been widely observed that the volatile solids (VS)-based methane production rate (ml CH4 gVSfeed-1 day-1) tends to

decrease as the TS increase in SS-AD, especially when using cellulosic biomass as a substrate. Abbassi-Guendouz et al.

(2012) investigated the effects of TS on AD by using cardboard as a substrate, and found that the VS based methane

production rate continued to decrease when the reactor TS increased from 10% to 35%. Similarly, Motte et al. (2013) used

wheat straw as a substrate to study the TS effect on SS-AD, and their results also showed a declining VS-based methane

production rate as TS increased from 15% to 25%. Many other studies have reported the same observation when using

various types of cellulose containing substrates such as cardboard, paper, municipal solid waste, cotton stalks, rice straw,

and others (Fernandez et al., 2010; Fujishima et al., 2000; Le Hyaric et al., 2012; Li and Wang, 2011; Pommier et al.,

2007; Radwan et al., 1993; Ramos et al., 2012).

Apparently, the effect of TS on SS-AD is two-faceted. If not carefully controlled, its adverse effect may offset the

expected advantages of SS-AD. Therefore, an improved understanding of the fundamental mechanism that has

compromised methane production rate by the effect of TS content is essential for optimizing the SS-AD process.

Traditional perceptions derived from L-AD studies have considered either hydrolysis or methanogenesis as the possible

rate-limiting step that constrains the methane production rate (Cirne et al., 2007; Vavilin et al., 2008). Recent SS-AD

studies showed that, when feeding recalcitrant cellulosic biomass as a substrate, if enough inoculum was provided, there

was very little volatile fatty acids (VFA) accumulation or pH drop, suggesting it is the cellulosic biomass hydrolysis that

has constrained SS-AD reactions (Abbassi-Guendouz et al., 2012; Bollon et al., 2011; Li and Wang, 2011; Zhao et al.,

2014). In an attempt to fit SS-AD experimental data into the L-AD-based Anaerobic Digestion Model No. 1 (ADM1),

Abbassi-Guendouz et al. (2012) found it is necessary to lower the first-order hydrolysis coefficient used in the model to

mathematically explain the reduced methane production rate of cardboard at elevated TS content. The mechanism behind

this reduced rate, however, was not elucidated.

Currently, limited information can be found about the compromised hydrolysis rate under high TS in AD process

2
(Vavilin et al., 2008), although it has been commonly observed in studies of enzymatic hydrolysis of cellulosic biomass for

ethanol production (Ballesteros et al., 2002; Cara et al., 2007; Ingesson et al., 2001; Kristensen et al., 2009; Lu et al.,

2002; Ramachandriya et al., 2013; Schwald et al., 1989). Kristensen et al. (2009) proposed that this phenomenon is an

“intrinsic or generic” effect of enzymatic hydrolysis at increased TS content. The inhibition to enzyme adsorption as a

result of excessive sugar accumulation could be responsible for the decreased hydrolysis rate at high TS content during

enzymatic hydrolysis (Kristensen et al., 2009).

Although this sugar inhibited hydrolysis in high TS content sounds like a plausible explanation to the existing SS-AD

perplexity, some key components are still missing. A major difference between enzymatic hydrolysis and SS-AD is that

the latter contains large amount of sugar consumers to quickly remove hydrolytic products upon their production,

especially when hydrolysis is a rate-limiting step. Only when a barrier exists that prevents microbial access to sugars,

could such a sugar accumulation phenomenon be manifested in an SS-AD system. Bollon et al. (2011) employed ADM1

in the examination of a hydrolysis-limited SS-AD system, and reached the conclusion that the Monod half-saturation

coefficient was always larger when the TS content was higher, indicating increased mass diffusion resistance in the dry

media of SS-AD. In line with their prediction, a recent measurement did show that the mass diffusion coefficients in SS-

AD were two orders of magnitude below the normal range in L-AD (Bollon et al., 2013).

Although the extent of internal mass diffusion resistance is a primary difference between SS-AD and L-AD, this factor

has not yet been considered in traditional modeling system such as ADM1. The objective of this study was to develop an

SS-AD tailored model to interpret the effect of TS content on the methane production rate, based on the hypothesis that

limited mass diffusion in SS-AD causes hydrolysis inhibition by accumulation of hydrolytic products such as sugar.

MATERIALS AND METHODS

PHYSICAL PROCESS AND ASSUMPTIONS
The model for this study was developed for the AD of cellulosic biomass in a batch bioreactor, with feedstocks and

inoculum completely premixed before loading. It was assumed that the inoculum, upon complete mixing with the

substrate, dispersed into many pinpoint microflora that were evenly distributed in the sludge bed (Figure 1). For

mathematical simulation, each microflora was assumed to be a small sphere enclosed in a substrate layer, with each thin

substrate layer in contact with several other substrate layers in a three dimensional sludge bed. These spheres were at a

micro-scale and the void in between was neglected. During AD, each microflora releases extracellular hydrolytic enzymes

into the surrounding substrate layer and convert cellulose and hemicellulose into sugars. The highest sugar concentration

was assumed to be at the outer surface of the substrate layer, and the lowest concentration was assumed to be inside the

3
microflora due to sugar consumption by the microbes. By the nature of mass diffusion, these sugars can only diffuse

towards the microflora inside due to the concentration gradients. Thus, a “watershed”, or more precisely, a “sugarshed”,

forms in the substrate layer, allowing sugars to flow into each microflora (Figure 1).

Biogas outlet

Microflora
X
A
Substrate layer
XF
V
Figure 1 Schematic illustration of the SS-AD model hypothesis

While it was hard to measure the diameter of these microflora, a typical 10-100 µm range was selected to represent the

syntrophic microflora formed in a well-mixed anaerobic digester (Thiele et al., 1988). According to the mass diffusion

theory, a diffusion boundary should exist at certain distance to the surface of each microflora, and no substance can diffuse

to the microflora beyond this boundary. The thickness of this diffusion boundary layer may range from 10 to 1000 µm in a

well-mixed bioreactor, depending on hydrodynamic conditions (Casey et al., 2000), thus in this study, the substrate layer

was thin enough (10-100 µm) to be within the range of this diffusion boundary layer. In addition, the cellulase released

from microflora was assumed to be evenly distributed in the substrate layer. Although a cellulase concentration gradient

probably exists in the substrate layer, limited research on cellulase diffusion in solid media was found in the literature;

therefore, this approximation was made.

This model was developed based on the premise that hydrolysis is the rate-limiting step in the AD process, thus

situations of acidification in overloaded reactors will not be included in this model. Other steps in AD reaction, i.e.

acidogenesis, acetogenesis, and methanogenesis, were combined into a single step. This combined step occurs fast enough

so that very few soluble monomers remain in the microflora. The microflora were assumed to be densely packed, with

synergic microbial communities living in close affinity, to make AD thermodynamically favorable (Schink, 1997). The

maximum allowable distance between syntrophic communities in AD is 1-2 µm (De Bok et al., 2004; Ishii et al., 2005;

McCarty and Smith, 1986). With all necessary microbes packed together, each viable microflora was assumed to function

as a discrete micro-reactor, which would expand across the substrate layer until merging with adjacent micro-reactors. The

endogenous activity of the population was neglected, and consequently no biomass decay was considered. In addition, the

existence of hydrolysis inhibition due to hydrolytic products accumulation was considered in this model development,

which was one of the major differences between this study and the previous ones.

4
MATHEMATICAL MODEL DEVELOPMENT
1) Biological reaction model
A first-order kinetics was assumed for cellulosic biomass hydrolysis under the circumstance without inhibition, which

can be written in Equation 1 according to ADM1 (Batstone et al., 2002),

R h = k h XF (1)

in which Rh is the hydrolysis rate; kh is the first-order hydrolysis coefficient; XF is the volatile cellulosic substrate

concentration in the substrate layer (Figure 1).

The hydrolysis inhibition was defined as,

R i = −k i S (2)

in which Ri is the hydrolysis rate reduction due to the sugar inhibition; ki is the hydrolysis inhibition coefficient; S is the

sugar concentration in substrate layer (Figure 1). This assumption is supported by the observation that in SS-AD, the

reduction of methane production rate under high TS content was be mainly due to the reduction of hydrolysis rate

coefficient (Abbassi-Guendouz et al., 2012). According to the studies about hydrolysis rate coefficient in enzyme

hydrolysis of cellulose during ethanol production, accumulated evidence showed that when sugar concentration was high,

the activity of hydrolytic enzyme would be inhibited (Hodge et al., 2008; Kristensen et al., 2009; Modenbach and Nokes,

2013). Although the exact kinetic equation describing this phenomenon has not yet been put forward, all experimental

results so far point to an inverted linear relationship between the sugar concentration and the hydrolytic enzyme activity

(Kristensen et al., 2009; Roberts et al., 2011).

The sugars diffused into the microflora were assumed to be utilized according to Monod equation (Equation 3).

µmax S ′ XM (3)
Ru =
(KS +S')Y∆M/S′

in which Ru is the sugar utilization rate; µmax and Ks are the maximum specific growth rate and the half-saturation

coefficient, respectively; Y∆M/S′ is the growth yield; S' is the sugar concentration on microflora surface. Since it is

unrealistic to experimentally determine the concentration of methanogens in a microflora, the VS concentration in

inoculum, namely XM, was used for proximity. Based on the premise that hydrolysis is the rate-limiting step, S' should be

insufficient for microbial utilization, therefore, Ks >> S' in Equation 3. Thus, Equation 3 becomes

µmax S ′ XM (4)
Ru =
KS Y∆M/S′

2) Mass diffusion model

Sugar diffusion from the substrate layer to the microflora can be predicted with Fick’s law in Equation 5,

De A (5)
Rd = (S − S ′ )
LV

5
 in which Rd is the sugar diffusion rate; De is the effective mass diffusion coefficient of sugars. Here it was assumed that

the sugar is glucose; L is the substrate layer thickness; A is the microflora surface area; V is the volume of the substrate

layer; S is the upstream sugar concentration in the substrate layer; S' is the downstream sugar on the microflora surface

(Figure 1).

Although the effective diffusion coefficient of a substance in gases and liquids can be successfully predicted by

theories, the effective diffusion coefficient in solid-like materials, such as SS-AD digestate, has a wide range, which may

differ by a factor of more than 1010 (Masaro and Zhu, 1999). Because there is no model currently available for predicting

the effective mass diffusion coefficient in SS-AD, in this study, an empirical stretched exponential equation was employed

for De estimation (Equitation 6), which was created for polymeric substances (Masaro and Zhu, 1999), and was

tentatively assumed to be applicable to cellulosic biomass.

ν
De = D ∙ e−αC (6)

in which C is the concentration of diffusion obstacles, namely C = XF/αF in this study (αF is the organic fractions in the

TS of substrate), and its unit is in g/L; α and ν are two scaling parameters.

3) Mass balance model

Referring to the schematic model in Figure 1, the feedstock-to-microbe (F/M) ratio (n) can be estimated with Equation

7. The F/M ratio is represented by the ratio of VS in substrate to that in the inoculum, and r is the microflora radius (Figure

1).

4 (7)
π�(L+r)3 -r3 �XF
n= 3
4 3
πr XM
3

Likewise, the TS content (XTS) can be calculated according to Equation 8 by summing up the TS contributed by

substrate and inoculum, in which αF and αM are the organic fractions in the TS of the substrate and inoculum, respectively.

4 4 XF 4 XM (8)
π(L+r)3 XTS = π�(L+r)3 -r3 � + πr3
3 3 αF 3 αM

Accordingly, the substrate layer thickness and the TS content in the substrate layer can be derived in Equation 9 and 10

from Equation 7 and 8, respectively,

1/3
XM (αM n + αF ) (9)
L=� � r−r
XTS αF αM

XM XTS αM αF n (10)
XF =
XM αM n − XTS αF αM + XM αF

4) Pseudo-steady state model

Because biogas production, as a function of digestion time in AD, produces a sigmoid-curve, the maximum methane

6
production rate on this curve can be estimated by using the modified Gompertz equation in Equation 11 (Zwietering et al.,

1990):

(R CH4 )max ∙ e ∙ (λ − t) (11)

CCH4 (t) = (CCH4 )max ∙ exp �− exp � + 1��
(CCH4 )max

in which CCH4(t) is the accumulative methane production at time t; (CCH4)max is the maximum methane production

potential; (RCH4)max is the maximum methane production rate; λ is the lag phase time; and t is the digestion time.

When hydrolysis is the rate-limiting step, it was assumed there is a pseudo-steady-state, at which the concentration of

all intermediate compounds do not change with time, namely

dS (12)
=0
dt

dS′ (13)
=0
dt

The combination of Equations 1, 2 and 5 gives the change of S as,

dS (14)
= Rh + Ri − Rd
dt

Likewise, the combination of Equations 4 and 5 gives the change of S′ as,

dS ′ (15)
= Rd − Ru
dt

Solving Equations 12 to 15 for S′ derives Equation 16,

k h XF (16)
S′ =
k VL µ X
k i + � i + 1� max ′ M
De A Y∆M/S K s

Assuming the maximum methane production rate was achieved with negligible changes in initial substrate and

inoculum concentration, namely XFo and XMo, the maximum methane production rate (R CH4 )max can be calculated by

substituting Equation 16 into Equation 4, and multiply a methane yield coefficient, namely Y∆CH4/∆S’ (Equation 17).

k h XF o Y∆CH4/∆S (17)
(R CH4 )max =
k i K s Y∆M/S′ k i LV
+ +1
µmax XM o De A

The XFo and L can be estimated from Equations 9 and 10. Along with other parameters from Table 1, Equation 17 can

be used to predict (RCH4)max for hypothesis verification against experimental data. The other unknown parameters, such as

ki, α, and ν, were regressed from experimental data using Matlab R2012b.

Table 1 Modeling parameters

Symbol Description Value Unit References
D Glucose diffusion coefficient at 37oC 9.8×10-6 cm2 s-1 (Hobbie, 2007)
µmax Maximum specific growth rate 8 d-1 (Rosen and Jeppsson, 2006)
Ks Half saturation coefficient 0.14 g L-1 (Rosen and Jeppsson, 2006)
kh Hydrolysis coefficient 10 d-1 (Rosen and Jeppsson, 2006)

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 Y∆M/∆S’ Microbial growth yield 0.04 g g-1 (Rosen and Jeppsson, 2006)
YCH4/∆S’ Methane yield 373 mL g-1 This study
XM Volatile inoculum concentration 124.46 g L-1 This study

AD EXPERIMENTAL PROCEDURE
1) Substrate and inoculum
Corn stover was collected from a farm operated by the Ohio Agriculture Research and Development Center (Wooster,

Ohio, USA). Before use, the corn stover was dried at 40°C in a drying oven (VWR/Sheldon Manufacture Inc., Cornelius,

OR, USA) for 24 hours to a moisture content of less than 10% and then ground to pass a 12.7 mm sieve (Mighty Mac,

MacKissic Inc., Parker Ford, PA, USA). L-AD effluent obtained from a mesophilic anaerobic digester (Akron, OH, USA)

fed with municipal wastewater sewage sludge was used as inoculum. The L-AD effluent was dewatered by centrifuge to

increase the TS from 9.0% to 14.9%. Characteristics of the corn stover and inoculum are shown in Table 2. The L-AD

effluent was incubated at 37±1 °C for 2 days to activate inoculum prior to use.

Table 2 Characterization of corn stover and inoculum

Parameters Corn stover Inoculum
Total solids (%) 97.3 ± 0.1 14.9±0.5
Volatile solids (%) 92.5 ± 0.2 9.7±0.2
Total carbon (%) 43.6±1.1 4.3±0.3
Total nitrogen (%) 0.5±0.0 0.6±0.0
Carbon to nitrogen (C/N) ratio 87.2±3.7 7.2±0.3
Cellulose (%) 44.5±0.0 ND
Xylan (%) 24.1±0.5 ND
Total ammonia nitrogen (gN/kg) ND 3.7±0.0
pH ND 8.8±0.1
Alkalinity (g CaCO3/kg) ND 14.4±0.5
Total volatile fatty acid (g/kg) ND 4.9±0.0
ND – not determined; numbers are mean ± standard error.

2) Batch mode anaerobic digestion

Anaerobic bottles (250 mL) were loaded with mixtures of corn stover and inoculum at a fixed F/M ratio = 2 (VS basis)

and different TS contents as shown in Table 3. Deionized water was added into each bottle to adjust the TS content from

1% to 28%. These bottles were connected to a multi-channel anaerobic respirometry system (Micro-Oxymax, Columbus

Instrument Inc., Columbus, OH, USA), and incubated at 37±1 °C in a water bath shaking at 70 rpm for 21 days. Biogas

production (CH4, CO2, H2 and H2S) from each bottle was real-time measured and recorded by computer. (RCH4)max was

estimated according to Equation 11.

Table 3 Characterization of the anaerobic bottles prepared for respirometry study

# TS Corn Stover Effluent Water Alkalinity Initial pH
% g g g mgCO3/kg
1 28 21.81 112.19 0.00 12.56 8.86
2 26 21.32 109.66 10.08 11.66 8.96
3 24 19.77 101.67 20.24 10.76 8.99
4 22 18.03 92.74 30.21 9.87 9.05
5 20 15.77 81.08 38.74 8.97 8.99
6 18 15.27 78.51 52.10 8.07 8.96
7 16 19.56 100.58 90.10 7.18 8.99
8 14 17.16 88.27 105.43 6.28 8.95
9 12 13.92 71.62 114.07 5.38 8.91
10 10 12.50 64.27 123.17 4.82 8.94
11 8 10.02 51.52 138.82 3.86 8.88
12 6 7.50 38.57 153.93 2.89 8.66
13 4 5.03 25.85 170.22 1.93 8.56
14 1 1.25 6.43 192.49 0.48 8.96

8
3) Analytical methods
TS and VS contents of corn stover and inoculum were analyzed according to standard methods (APHA, 1998). Total

carbon and nitrogen contents were determined by an elemental analyzer (Vario Max CNS, Elementar Americas, Mt.

Laurel, NJ, USA), and were used to calculate the carbon to nitrogen (C/N) ratio. The pH was measured by a pH meter at

20°C (Oakton pH 11 Standard Portable Meter, Oakton Instrument, Vernon Hills, IL USA). Alkalinity was measured

following a titration procedure (McGhee, 1968) using an auto-titrator (Mettler Toledo, DL22 Food & Beverage Analyzer,

Columbus, OH, USA). Samples for alkalinity measurement were prepared by diluting five grams of digestate with 50 mL

of deionized water, mixing for 2 minutes, and then filtering through four layers of cheese cloth.

RESULTS
MODEL VERIFICATION WITH EXPERIMENTAL DATA FROM LITERATURE
This study attempted to mathematically interpret the effect of TS content on the methane production rate, based on the

hypothesis that limited mass diffusion in SS-AD will cause hydrolysis inhibition by accumulation of hydrolytic products,

i.e. sugar. This hypothesis was first verified using experimental data from Abbassi-Guendouz et al. (2012), who studied

the AD of cardboard at F/M=20, and determined the maximum methane production rate (RCH4)max at eight TS

concentrations ranging from 10% to 35%. Their results showed the (RCH4)max values, calculated on a VS basis, decreased

continuously as TS increased. However, theoretically, the reaction rate is usually calculated based on per mixed liquor. In

order to substitute these data into the theoretical model developed in this study, their units were converted based on per

mixed liquor mass. The simulated results in Figure 2 show that the maximum methane production rate per mixed liquor

mass ((RCH4)max) increased with TS from 10% to 20%, and then decreased from 20% to 35%. The model prediction agreed

well with this bell-shaped curve with R2 = 0.87. This close correlation between experimental data and model prediction

provided strong support to the mass diffusion-caused hydrolysis inhibition hypothesis proposed in the model development.

This model also satisfactorily fit data from other literature (Fernandez et al., 2010; Le Hyaric et al., 2012; Li and Wang,

2011). However, one problem in using these literature data is their limited sample size for model verification. The eight

data points in the study by Abbassi-Guendouz et al. (2012) were the maximum found in the literature.

9
Figure 2 Simulation of the TS effect on maximum methane production rate with data from study by Abbassi-Guendouz et al. (2012). The model
prediction is shown by the line at αF = 0.77, αM = 0.50, ki = 9.25×107 d-1, α = 2.45×10-5, ν = 2.01, R2 = 0.87

MODEL VERIFICATION WITH EXPERIMENTAL DATA FROM THIS STUDY

To improve confidence in the model fitness, experimental data with fourteen data points were obtained from a multi-

channel anaerobic respirometry system using corn stover as the cellulosic substrate, with the TS ranging from 1% to 28%

and F/M ratio of 2 (Table 3). The model prediction, as shown in Figure 3, provided adequate mathematical description of

the experimental results with R2 = 0.92. The (RCH4)max kept increasing with TS from 1% to 15%, and then gradually

dropped when TS further increased from 15% to 28%. This satisfactory model fitness further verified the adequacy of the

proposed model in describing the effect of TS content on SS-AD performance.

Figure 3 Simulation of the TS effect on maximum methane production rate with data obtained from respirometry study. The model prediction
is shown by the line at αF = 0.92, αM = 0.50, ki = 9.32×104 d-1, α = 0.03, ν = 0.94, R2 = 0.92

MODEL VERIFICATION WITH STATISTICAL DATA FROM LITERATURE

By means of a three level Box-Behnken experimental design, Motte et al. (2013) determined the statistical relationship

between TS, particle size, and the accumulative methane production in AD of wheat straw. Statistical models have been

commonly used as a mathematical tool to assess the stochastic relationship between variables without understanding the

mechanism in-depth. Therefore, an examination of the theoretical model developed in this study against a statistical model

derived from another source may provide an unbiased, informative reference for model validation. The data obtained

from Motte et al. (2013) at three substrate particle sizes were also fit into Equation 17. Since only the accumulative

methane production was reported in their study, it was used to calculate the average methane production rate and assumed

10
it to be proportional to the value of (RCH4)max. As can be seen, the data produced from the two disparate models agreed

well with each other (Figures 4 A to C). Again, bell-shaped curves are shown in Figures A to C with the peaks at TS of

between 15% and 20%. It should be noted that Equation 17 was derived from a theoretical hypothesis, while the data by

Motte et al. (2013) was determined by statistical prediction. The conformity between the two supports the rationality of the

theoretical model developed in this study.

A B

Figure 4 Simulation (line) of the TS effect on average methane production rate with statistical data predicted by Motte et al. (2013) for: A)
coarse wheat straw digestion at r = 25 µm, αF = 0.92, αM = 0.50, ki = 8.26×107 d-1, α = 2.44×10-4, ν = 1.66, F/M = 42, R = 0.95; B) medium
wheat straw at r = 25 µm, αF = 0.94, αM = 0.50, ki = 3.02×107 d-1, α = 3.70×10-3, ν = 1.26, F/M = 42, R = 0.99; C) fine wheat straw digestion at r
= 25 µm, αF = 0.92, αM = 0.50, ki = 1.45×108 d-1, α = 7.78×10-6, ν = 2.29, F/M = 32, R2 = 0.99

DISCUSSION
As can be seen in Figures 2 to 4, TS content had a two-faceted effect on methane production rate. A theoretical model

assuming mass diffusion-caused hydrolysis inhibition can be used to interpret this two-faceted effect. In cellulosic

biomass-fed L-AD, organic solids provide the primary energy source for microbial growth, and thus contribute to methane

production following the Monod equation in Equation 4, which is embodied in the left-hand side of the bell-shaped curve

in Figures 2 to 4. However, in SS-AD, the TS content started to manifest its inhibitory effect by increasing mass diffusion

resistance as described in Equation 2, and resulted in the right-hand side of the bell-shaped curve in Figures 2 to 4. This

two-faceted effect of TS should exist in all AD systems, but the dominant facet may mainly depend on the magnitude of

TS. Results from Figures 2 to 4 suggest that within a range of TS between 15% and 20%, there was a turning point where

11
the methane production rate of AD turned from increasing as TS increased, to decreasing. This turning point is the

optimum TS content for the most cost-effective methane production from SS-AD of cellulosic biomass.

Theoretically, mass diffusion resistance itself does not directly constrain the methane production rate, thus other

possibilities, such as inhibition, should be considered. The reason is that in a hydrolysis-limited AD process, all

hydrolyzed sugars have one destination, that is, being converted into methane. As a result, under strong mass diffusion

resistance, a steeper mass diffusion gradient would be established to accelerate the transfer of hydrolytic products to

microbes. Hence, the methanogenesis rate should synchronize with the hydrolysis rate regardless of the magnitude of mass

diffusion limitation when the reaction proceeds at a steady state. Since this was not observed in experiment, the adverse

effect of high TS content on SS-AD performance must come from the reduced hydrolysis rate in the first place when

hydrolysis is the rate limiting step.

Results shown in Figures 2 to 4 suggests that incorporating a mass diffusion-caused product inhibition mechanism

provides a reasonable explanation for the two-faceted effect of TS content on AD. There are many hydrolytic products

known for causing hydrolysis inhibition (Vavilin et al., 2008). Without timely removal of these inhibitors by downstream

consumers, the hydrolysis rate will be compromised. As a result of the increasing TS content, a steeper gradient of

hydrolytic products, such as sugars, may cause hydrolysis inhibition in substrate layers.

The extent of such a product accumulation can be represented by (S/S′) in Equation 18 which is derived from Equations

12 to 16. S/S′ is the ratio of substrate concentration at the substrate layer and in the microflora.

S L V XM µmax (18)
= 1+
S′ K s Y∆M/S′ De A

S/S′ profiles were calculated for microflora radii (r) of 10, 50, and 100 µm and plotted against diffusion distances (L)

of up to 100 µm using three different effect diffusion coefficient De (Figure 5). To our knowledge, the work by Bollon et

al. (2013) was the only study so far that has determined the likely magnitude of De in SS-AD, which is two orders lower

than D in water. One possible explanation is that most of the diffusion takes place in the capillary structure of the solid

substrate, which reduced the diffusion rate compared with in the bulk solution (Bollon et al., 2013). The capillary structure

of the solid substrate at TS of between 15% and 20% may have affected the internal porosity and tortuosity, and thus the

diffusion coefficient. Therefore, De = 10-5, 10-6 and 10-7 cm2 s-1 were used in Equation 18 for simulation as they are the

most probable sugar diffusion coefficients in SS-AD at 37oC. At least three trends can be seen in Figures 5: i) the decline

in De significantly contributed to hydrolytic product accumulation in terms of S/S′ in AD, e.g. about 1000 times more

product would be accumulated in SS-AD with De = 10-7 cm2 s-1 as compared to L-AD with De = 10-5 cm2 s-1 at the same

diffusion distance (L); ii) the extent of product accumulation quadractically increased with the diffusion distance, e.g. as L

12
doubles, S/S′ quadruples; iii) the size of microflora also has a significant influence on S/S′, especially when r is increased

from 10 to 50 µm, i.e., a larger microflora radius provides more surface area for sugar influx and thus mitigates the extent

of S/S′. With these three effects combined, S/S′ ranging from 104 to 105 can be accumulated at a distance of merely 50 µm

from the microflora surface at De = 10-7 cm2 s-1 (Figure 5 A to C). Thus, it can be seen that severe hydrolytic product

inhibition can be expected in SS-AD due to mass diffusion limitation at high TS contents.

Figure 5 Relationship between S/S′ ratio and diffusion distance L at: A) r = 10 µm, B) r = 50 µm, C) 100 µm, and De = 10-5 cm2 S-1 ( ),
De = 10-6 cm2 S-1 ( ), De = 10-7 cm2 S-1 ( ), respectively

Results shown in Figures 5 also indicates that due to the aggravated product inhibition at distances further from the

microflora, the effective hydrolysis may only occur within several micrometers next to the microflora surface, which

provides an explanation for the observation that effective hydrolysis only occurs adjacent to hydrolytic microflora (Wang

et al., 2011). That being said, the effective amount of substrate available for hydrolysis action should be much less than the

XF value used in Equation 17. Consequently, an SS-AD system with a hydrolysis inhibition zone is believed to have a

methane production rate lower than an L-AD system that does not have the inhibition zone.

Given the importance of microflora radius and the diffusion distance in determining the extent of product accumulation,

it is necessary to estimate their possible range in an SS-AD system. Despite little available information in the literature

regarding this aspect, from Equation 9, the relative ratio of L/r in SS-AD can be estimated. According to Figure 6, on one

hand, the L/r ratio increases with F/M ratio, e.g., L/r at F/M = 20 is four times of that at F/M = 2 when TS = 10%; on the

13
other hand, L/r decreases with an increase in TS content because of layer compression in a crowded environment. Thus, it

can be seen that L/r should range from 1.3 to 2 in an SS-AD system (Figure 6), which means, if r ranges from 10 to 100

µm, L would be 13 to 200 µm. The striking order of magnitude of the S/S′ accumulation within this range can be

conceived by referring to Figures 5 A to C at De = 10-7 cm2 s-1. In line with the prediction based on Figure 6, the hydrolysis

inhibition coefficient, ki, simulated from Figures 2 and 4 at F/M = 20 ~ 42 are at least three orders higher than the value

simulated from Figure 3 when F/M = 2.

Figure 6 Relationship between L/r ratio and TS at n ranging from 2 to 20, and Xm =124.46 g/L, αM = 0.5, αF = 0.92

The goals for using high TS cellulosic feedstocks for ethanol production are similar to those of SS-AD, that is, to

decrease water consumption and reduce equipment and energy costs. Cellulosic ethanol production has encountered the

same problem of severe hydrolysis inhibition by end-products such as cellubiose and glucose. It has been proposed that

the end-product inhibition of cellulase is rate-limiting for lignocellulose hydrolysis under high TS content (Kristensen et

al., 2009). While it is easy to detect sugar inhibition in the scarification process where pure cellulases are the only catalyst,

the measurement of sugar gradients at a micro-scale in SS-AD could be experimentally challenging. Bollon et al. (2013)

have speculated the mass transfer limitation could cause strong substrate accumulations in SS-AD, leading to partial

inhibitions and reducing the overall process efficiency. This study has provided quantitative evidence for the existence of

such a mass diffusion-caused hydrolysis inhibition zone in SS-AD. It must be realized that, even though this model

assumption was limited to a completely premixed system, which may not readily extend to an inhomogeneous system as

such as a landfill, usually in a system with high TS content and poor agitation, sugars produced from hydrolysis would

have to migrate through even longer distances within the digestion medium to become available to microflora, which

could further limit the overall SS-AD reaction rate.

14
CONCLUSION
The influence of TS content on SS-AD performance is two-faceted. There is a TS content threshold between 15% and

20% for AD of lignocellulosic biomass, below which the methane production rate tends to increase with TS, and vice

versa when exceeding it. Incorporating a mass diffusion-caused hydrolysis inhibition mechanism into the AD model was

able to provide a reasonable interpretation of this two-faceted effect of TS content in SS-AD part. The proposed model is

capable of giving a theoretical basis for phenomena manifested in experimental measurement and statistical prediction.

The proposed mechanism is important for understanding the performance deterioration in SS-AD with increasing TS.

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