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Kinetics of anaerobic treatment: A critical review

a b
S. G. Pavlostathis & E. Giraldo‐Gomez
a
School of Civil Engineering, Georgia Institute of Technology, Atlanta, GA, 30332
b
Department of Civil Engineering, University of Massachusetts, Amherst, MA, 01003
Version of record first published: 09 Jan 2009.
To cite this article: S. G. Pavlostathis & E. Giraldo‐Gomez (1991): Kinetics of anaerobic treatment: A critical review, Critical
Reviews in Environmental Control, 21:5-6, 411-490
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Critical Reviews in Environmental Control. 21(5,6):411-490 (1991)
Kinetics of Anaerobic Treatment: A

Critical Review
S. G. Pavlostathis
School of Civil Engineering, Georgia Institute of Technology, Atlanta, GA 30332
E. Giraldo-Gomez
Department of Civil Engineering, University of Massachusetts, Amherst, MA 01003
Downloaded by [University of Arizona] at 11:08 03 January 2013
ABSTRACT: The fundamentals of microbial kinetics and continuous culture models are presented.
The kinetics of the anaerobic treatment processes are reviewed recognizing that anaerobic degra-
dation of complex, polymeric organic materials is a combination of series and parallel reactions.
Such reactions include hydrolysis, fermentation, anaerobic oxidation of fatty acids, and methan-
ogenesis. The intrinsic rates of each step are reviewed and literature data summarized. Whenever
possible, available kinetic information is summarized on the basis of substrate composition (such
as carbohydrates, proteins, and lipids). The effect of temperature and inhibitors on the intrinsic
kinetic rates is discussed. Stoichiometric and bioenergetic considerations are reviewed. Mass transfer
limitations (both external and internal) associated with biofilms and microbial agglomerates, in
general, and their effect on the intrinsic kinetic rates are presented. Areas requiring further research
are identified.
KEY WORDS: kinetics, anaerobic digestion, hydrolysis, methanogenesis, mass transfer, bioen-
ergetics, carbohydrates, protein, lipids.
I. INTRODUCTION
Anaerobic digestion is one of the oldest biological wastewater treatment

processes, having first been used more than a century ago.1 With the development
of digester heating and mixing, anaerobic digestion has become the most common
method of sludge stabilization. Because of the growing emphasis on energy
conservation and recovery as well as other environmental concerns related to land
disposal of wastewater sludges, anaerobic digestion is expected to continue to
play a major role in municipal sludge processing.2 Other complex feedstocks to
which the anaerobic digestion process has been applied include agricultural wastes
(e.g., plant residues, animal waste) and food-processesing wastewaters, all of
which are considered concentrated wastes (i.e., high content of biodegradable
1040-838X/91/S.50
© 1991 by CRC Press, Inc.
411
organics). Recognition of the advantages of anaerobic processes, over those of
aerobic processes, has also led to the development of new anaerobic process
configurations capable of treating medium and low strength soluble and colloidal
wastes (e.g., municipal wastewater).3 Advances in process engineering have also
been supported by recent developments in the chemistry, biochemistry, and mi-
crobiology of anaerobic processes.
Process kinetics play a central role in the development and operation of
anaerobic treatment systems. Based on the biochemistry and microbiology of the
anaerobic process, kinetics provide a rational basis for process analysis, control,
and design. In addition to the quantitative description of the rates of waste uti-
lization, process kinetics also deal with operational and environmental factors
affecting these rates. A sound knowledge of kinetics allows for the optimization
of performance, a more stable operation as well as better control of the process.
The objective of this paper is to review the available kinetic information in

order to better understand the various steps involved in the anaerobic degradation
of organic substrates and their conversion to methane. Whenever possible, the
intrinsic rates of each step as well as the effect of temperature and inhibitors on
these rates are discussed. Mass transfer limitations and their effect on the intrinsic
kinetics are presented. Areas requiring further research are also identified.
The anaerobic degradation of complex, particulate organic material has been
described as a multistep process of series and parallel reactions (see Figure I). 4 " 9
First, complex polymeric materials such as polysaccharides, proteins, and lipids
(fat and grease) are hydrolyzed by extracellular enzymes to soluble products of
small enough size to allow their transport across the cell membrane. These rel-
atively simple, soluble compounds are fermented or anaerobically oxidized to
short-chain fatty acids, alcohols, carbon dioxide, hydrogen, and ammonia. The
short-chain fatty acids (other than acetate) are converted to acetate, hydrogen
gas, and carbon dioxide. Lastly, methanogenesis occurs from carbon dioxide
reduction by hydrogen and from acetate.
The anaerobic digestion of complex organic materials can be divided into
seven subprocesses, following the reaction scheme suggested by Gujer and
Zehnder:8
1. Hydrolysis of complex, particulate organic materials

2. Fermentation of amino acids and sugars
3. Anaerobic oxidation of long-chain fatty acids and alcohols
4. Anaerobic oxidation of intermediary products (such as short-chain fatty
acids, except acetate)
5. Acetate production from carbon dioxide and hydrogen (homoacetogenesis)
6. Conversion of acetate to methane (aceticlastic methanogenesis)
7. Methane production by reduction of carbon dioxide by hydrogen
Anaerobic degradation of viable, biological solids (e.g., waste activated sludge,

algae) requires an additional conversion mechanism, namely, death and lysis of
412
COMPLEX POLYMERS
PROTEINS CARBOHYDRATES LIPIDS
HYDROLYSIS
AMINO ACIDS. SUGARS FATTY ACIDSw ALCOHOLS
FERMENTATION INTERMEDIARY PRODUCTS ANAEROBIC

(Propionate. Butyrate etc)
OXIDATION
ACETICLASTIC REDUCTIVE
METHANOGENESIS METHANE METHANOGENESIS
CARBON DIOXIDE
FIGURE 1. Reaction scheme for the anaerobic digestion of polymeric materials (numbers
indicate the bacterial groups involved, see text). (Adapted from References 8 and 9.)
viable cells before the organic material is hydrolyzed. This step is discussed later
in conjunction with the hydrolytic step.
The major groupings of bacteria and the reactions they mediate are as follows9
(1) fermentative bacteria; (2) hydrogen-producing acetogenic bacteria; (3) hydro-
gen-consuming acetogenic bacteria; (4) carbon dioxide-reducing methanogens;
and (5) aceticlastic methanogens (see Figure 1). Recently, the microbiology and
biochemistry of anaerobic processes have been reviewed and are not discussed
here.5-7-10-16
The nature and chemical composition of the materials used in anaerobic
digestion dictate the operative degradation subprocesses and the microbial groups
involved in the anaerobic conversion of these substrates. Table 1 shows the
chemical composition of various materials commonly used as substrates for an-
aerobic digestion. The major component of activated sludge is crude protein.
Meat-packing waste and primary sludge have a relatively high lipids content.
Lignin and hemicellulose are significant components of animal waste and mu-
nicipal refuse.
413
TABLE 1
Composition of Various Substrates Commonly Used for Anaerobic Digestion (% of Dry Matter)
Meat
Municipal packing Cattle Chicken
Component Primary municipal sludge Activated sludge Refuse waste manure manure
Volatile solids 79.7 73.5 75.0 59-75 79.0 81.6-82.1 92.0 72.0 76.0
Lipids 18.6 21.0 10.3 5-12 5.8 5.98-6.36 54.6 3.5 1.5
Cellulose 18.2 19.9" 32.2 7" 9.7" 35.0-37.0 — 17.0 28.3
Hemicellulose — — 2.5 — — 16.51 — 19.0 11.9
Lignin — — 13.6 — — — — 6.8 9.2
Crude protein 17.2 28.7 19.0 32-41 53.7 5.78-6.07 28.7 19.0 28.8
Volatile acids 3.5" 5.0 6.4° — — — — — —
Ash 20.3 26.5 25.0 25-41 21.0 17.9-18.4 8.0 28.0 24.0
Ref. 17 18 19 2 20« 21 22 23 24
Expressed as acetic acid.

Measured as total carbohydrates.
Total free fatty acids.
Includes lignin.
Biological sludge derived from laboratory units fed with totally soluble feed.
Difference between total carbohydrates and cellulose.
II. FUNDAMENTALS OF MICROBIAL KINETICS
A. Fundamental Relationships in Biological Growth Kinetics
The quantitative description of microbial growth kinetics has greatly advanced

since the development of the theory and application of the continuous flow cul-
tivation of microorganisms.25"27 As a result, biological treatment processes have
been successfully described by the theory of continuous cultivation of microor-
ganisms. Process kinetics have been used for the mathematical description of
both aerobic and anaerobic biological treatment processes.28-29
Biological growth kinetics are based on two fundamental relationships: growth
rate and substrate utilization rate. Homogeneous culture conditions (i.e., uniform
concentration of biomass and substrate) are assumed for the following discussion.
1. Growth Rate
If conditions are conducive to growth, the rate of microbial growth can be

expressed by the autocatalytic equation:30
(1)
f - •*
where X = microorganism concentration (M L~3); t = time (T); |x = (HX)(dXI
dt) = specific growth rate (T" 1 ).
Endogenous respiration, commonly defined as the self-destruction of biomass,
cell maintenance, prédation, and cell death and lysis are processes leading to a
decrease in cell mass. These processes are important in waste treatment systems,
especially anaerobic systems, since they usually operate at low specific growth
rates. To account for the effect of these processes on the net growth rate, a
microorganism decay rate is usually used for the modification of the growth
rate.28-29
where b = specific microorganism decay rate (T" 1 ).
2. Growth Yield and Substrate Utilization Rate
The relationship between the rates of microbial growth and substrate utili-
zation is as follows
415
dS (dX/dt)
(3)
~dt - - ~Y~
where Y = growth yield coefficient (M M" 1 ). Using the definition of |A, the
following equation is obtained:
^ . = » = U (4)
K
Xdt Y '
where: U = l/X(dS/dt) = specific substrate utilization rate (M M" 1 T" 1 ). When
\i = \L = maximum specific growth rate, then: {UY = I/m„ = k = maximum
specific substrate utilization rate (M M" 1 T" 1 ).
3. Effect of Substrate Concentration on the Microbial Growth Rate
The effect of the growth-limiting substrate (i.e., the essential nutrient) con-
centration on the rate of microbial growth has been described by various math-
ematical models31"34 However, the most widely used kinetic model is that of
Monod:31
where: (i, = maximum specific growth rate (T" 1 ); S = concentration of the

growth limiting substrate (M L~3); and Ks = half-velocity coefficient (i.e.,
substrate concentration at one-half maximum specific growth rate, M Lr 3 ). A
similarity exists between this model and the Michaelis-Menten equation for en-
zyme kinetics. The Monod equation was developed as a result of an empirical
analysis of bacterial growth data. The Monod equation expressed in terms of the
specific substrate utilization rate is
Equation 6 becomes zero-order for S >KS and first-order for S <KS.

By combining Equations 2 and 5 and setting dXIdt = 0, the equation for the
minimum substrate concentration (S^ for microbial survival (but not net growth)
is obtained:
bK
Ç - ' (l\
p, — b
When Contois33 applied the Monod model to fit data of continuous cultures of
416
Aerobacter aerogenes grown on defined media (glucose and succinic acid were
the carbon sources), the half-velocity coefficient was found to be proportional to
the influent substrate concentration (So), i.e., K, = a So, where a is a propor-
tionality constant (dimensionless). This led to a modification of the Monod equa-
tion as follows
By neglecting the microorganism decay coefficient, Contois derived the following

equation by incorporating the microorganism concentration (X) into the previous
equation:
(9)
BX + S
where um = p7(l + a) and B = a/Y(l + a).

Chen and Hashimoto35 used the Contois model (Equation 9) and derived the
following equation:
^ KS0 + (1 - K)S
where K = al(\ + a) = constant (dimensionless). Chen and Hashimoto35 used

pi = um. However, based on the Contois notation, pi = um only when a is very
small. Other kinetic models are presented in Table 2.
B. Continuous Culture Models
By use of the above presented fundamental kinetic relationships and applying

mass balances for the biomass and substrate, explicit equations have been de-
veloped for various reactor configuration. For the purposes of illustration, a
completely-mixed, constant volume reactor (CSTR) is considered here. Influent
microorganism concentration is considered negligible for the following discus-
sion. The steady-state effluent (and reactor) microorganism concentration is given
by the following equation regardless of the substrate utilization model:28
x
- e c
where X = microorganism concentration (M L~ 3 ); 0 = hydraulic retention time

(T); 6C = solids retention time (or mean cell residence time) (T); and So =
influent substrate concentration (M L~ 3 ).
417
TABLE 2
Kinetic Models Used in Anaerobic Treatment
First-order
kS . -dS ._ . So
p.
p
= — - b; ——- = kS; S = -—2;— v
(A)
So - S ' cff ' 1 + /c8c '
Grau et al.
= ¿ § _ b. ~dS = ¿ * § . e = So(1 + b9e)
Monod
_ h. ZEE- ¿xs . o _ W + fee.)

' dt YÍK. + S)1 e c (ti-/>)-i
Contois
^ BX+ S ' dt Y(BX + S)'
bec)
+ ¿ec) + ee(i/m - b) - 1
(D)
Chen and Hashimoto
= y,S S ÍLXS
M
' ~ /CS + (1 K)S ' =
/CS 0 + (1 - K ) S ' d t K X + YS'
(E)
A/oie: 5 is the effluent substrate concentration for a CSTR reactor at steady-state; k is the
maximum specific substrate utilization rate; Ks is the half-saturation constant (see
text for other terms).
For a CSTR at steady-state, the following expression holds true:
0 r = (YU - fe)-1 (12)
Combining Equations 6 and 12 yields:
«• - hg, - »r
418
When the inverse of 8C is higher than the maximum specific growth rate, washout
of the microorganisms will occur. This is the minimum value of 0C (i.e., 9™in)
and is defined as follows: 9C = 6™n when S = So, i.e., process failure occurs
at the minimum 8,. values. Based on Equation 13, the following expression is
obtained:
For cases where So > Ks, then:
¿]-> (15)
where 6"™ = limiting solids retention time (T). From the above equations, we
can conclude that the minimum solids retention time for a CSTR is determined
by the values of the kinetic constants Y, k, Ks, and b. Table 2 shows various
kinetic models used in anaerobic digestion studies. The usefulness of the Contois,
Chen and Hashimoto and Grau et al. models is that the predicted effluent substrate
concentration's) is a function of influent substrate concentration (So). This is an
improvement over the Monod model where S is independent of So. In essence,
the former two models explicitly account for the organic loading which has been
found to affect digester performance. However, in our view, the Contois model
is in essence the Moriod model except that K, is considered to be a function of
5"0. To prove this point, the equation for the effluent substrate concentration (5)
as given by both the Contois and the Chen and Hashimoto models (Equations D
and E in Table 2) can be reduced to the following equation when we substitute
the values of um, B, and K as defined by Contois and Chen and Hashimoto (see
above):
By using Ks = a So, the above equation reduces to the equation of effluent

substrate concentration based on the Monod model (Equation C in Table 2).
Several researchers have used the Contois-Chen and Hashimoto model. In our
view, the only benefit of the Contois model, i.e., the direct effect of So on 5,
can be dealt with a variable Ks in the Monod model without using unnecessary
changes of notation and introduction of new variables which lead to confusion
and misinterpretation of the K, and 5 0 relationship originally presented by Contois.33
C. Kinetics of Hydrolysis of Particulate Matter
The most commonly applied model for the description of the hydrolysis rate
419
in anaerobic systems is first-order with respect to the concentration of degradable
paniculate organic matter (F, M L" 3 ):
=
f
where kh = hydrolysis rate coefficient (T" 1 ). For a batch reactor, integration of
Equation 17 leads to:
F = Foe-1-' (18)
where Fo = concentration of degradable, particulate organic matter at t = 0 (M

L~ 3 ). For a CSTR at steady-state, the following equation is obtained:
F
-
where Fo, F = influent and effluent concentration of degradable, particulate

organic matter, respectively (ML" 3 ).
D. The Rate-Limiting Step Approach
As previously described, the anaerobic treatment of complex organics is a

multistep process. Most of the early attempts to kinetically describe the anaerobic
treatment process relied upon the so-called rate-limiting step approach. Generally
speaking, when a process is composed of a sequence of reactions, one step is
usually very much slower than the other steps. In this situation, as Hill36 has
stated: "The rate of product formation may depend on the rates of all the steps
preceding the last slow step, but will not depend on the rates of any of the
subsequent, more rapid steps." The last slow step in a sequence of reactions has
been called the rate-controlling, rate-limiting, or rate-determining step. 36
Lawrence29 proposed that in anaerobic digestion processes "the rate-limiting step
be defined as that step which will cause process failure to occur under imposed
conditions of kinetic stress." In the context of a continuous culture, kinetic stress
refers to the imposition of a continually reducing value of the solids retention
time until it is lower than its limiting value and results in washout of the micro-
organisms. In anaerobic treatment, washout-type failure leads to near cessation
of methane production, decreased COD destruction, and a build-up in the con-
centration of long- and short-chain fatty acids.29'37
This description of process failure is consistent with the hypothesis in which
one step is considered to be the limiting one. However, one must recall that steps
preceding the last slow step can influence the overall rate. Such is the case with
methane fermentation of particulate organic materials: the point at which washout
420
of methanogens occurs, considered by Lawrence as the " t r u e " process failure
point, is indeed influenced by preceding steps (e.g., hydrolysis of particulates).
The rate of hydrolysis determines, for a given retention time, the potential max-
imum substrate concentration possible for methanogens, which in turn determines
their maximum possible specific growth rate.
Even in cases where acidogenesis or methanogenesis are considered to be
limiting steps, hydrolysis may affect the overall process kinetics, a point too
often overlooked. T o illustrate the effect of the hydrolysis step on subsequent
fermentative steps, consider the effect of hydrolysis on the wash-out retention
time of acidogenic bacteria. Assuming first-order kinetics for the hydrolysis step,
the effective maximum substrate concentration available to the fermentation bac-
teria is as follows
_ F _
kh%e
(Fo, F, and kh as previously defined). By definition, process failure will occur

when S = iS0, i.e., S = Fo — F. If the Monod model is used for the kinetic
description of the fermentation step, by use of Equation C in Table 2, the process
failure for a CSTR reactor will occur when:
+ bec)
0C((A - b) - I 1 + kA
The minimum solids retention time can be estimated from the above analysis.
Figure 2 shows the values of 0™n as a function of kh for a case where (L = 0 . 4
d" 1 , b = 0.05 d " 1 , and for three values of FJKS: 5 , 10, and 20. From these
results, w e can conclude that the lower the value of kh, the higher the 0^", noting
that this effect is more pronounced at low ratios of FJK,. When kh is large, So
=* Fo and the true 0 ^ " can be calculated based on Equation 14. For example, for
the case presented here with FJK, = 10, for large kh, Qf" = 3.19 d. However,
when kh = 0.1 d ~ \ 0^™ = 4.05 d. In other words, the minimum solids retention
time is increased by approximately 27%.
In their effort to model anaerobic digestion kinetics, various investigators
have considered only some of the steps outlined in Figure 1 to be relevant to
their work. For example, O'Rourke 1 7 looked at steps such as hydrolysis, fer-
mentation, acidogenesis, and methanogenesis but his kinetic formulation was
based on the conversion of long- and short-chain fatty acids to methane. Others
have pointed out that the hydrolysis step is also very important. 18>38~41 It is quite
clear that the type of waste being digested (e.g., soluble v s . paniculate; or its
chemical composition) dictates which steps need be considered, thus explaining
why different investigators dealing with different wastes have arrived at quite
diverse model formulations. In anaerobic digestion, the rate-limiting step is related
to the nature of the substrate, process configuration, temperature, and loading
rate. 42
421
16.00
CO
>» 12.80
at
O
9.60 H
CC
CO
2 6.40 •
I
z
= 3.20 -
0.00
0.00 0.04 O.08 0.12 0.16 0.20
HYDROLYSIS CONSTANT (kh , d"1)
FIGURE 2. Effect of the hydrolysis rate constant on the minimum solids retention time at
different levels of the relative influent substrate concentration (F/K,) (biokinetic coefficients
used: jl = 0.4 d - 1 ; 6 = 0.05 d" 1 ).
III. STOICHIOMETRY AND BIOENERGETICS
The chemical composition of the organic matter subjected to anaerobic diges-

tion, along with the bacterial species involved, dictate the type and amount of
the products. When the chemical composition of the organic matter is known,
the following stoichiometric equation developed by McCarty can be used for the
overall conversion of the organic matter to methane:43-44
CJIJ)Jfe+-(2n + c - b - ^ - ^JH2O -> j CH4
-c-S4- d
4Vo.
20
(c - g
(< - I
422
where d = An + a — 2b — 3c; s = fraction of waste converted to cells; e =
fraction of waste converted to methane gas for energy (s + e = 1); CJIjOJJ,.
= empirical formula of waste being digested; CflnOJ<l = empirical formula of
bacterial dry mass (i.e., VSS).
Stoichiometric equations for the production of intermediates can also be
developed. McCarty43-45"47 has presented a procedure based on thermodynamic
and bioenergetic principles which allows the derivation of stoichiometric equations
as well as the estimation of microbial yields and specific substrate utilization
rates. In short, the energy released during respiration is converted to ATP energy
to be used for microbial synthesis. The energy required for the microbial synthesis
is broken into three parts: (1) free energy change from the conversion of the
electron donor (ed) to pyruvate (assumed to be a common intermediate for mi-
crobial synthesis); (2) free energy change for the reduction of the nitrogen source
to the ammonia level; and (3) free energy required to convert pyruvate and
ammonia to biomass. Accounting for the inefficiencies involved in the energy
conversions and applying an energy balance, the number of electron equivalents
of the electron donor converted for energy per electron equivalent of cells syn-
thesized (A) can be calculated. All balances are made using the electron equivalent
(eeq) as basis [one electron equivalent is equal to 8 g theoretical oxygen demand
(ThOD)]. Based on the value of A, the microbial yield coefficient (Y, g VSS/g
ThOD) can then be calculated. Figure 3 shows the effect of solids retention time
on the observed yield coefficient for various substrates and intermediates com-
monly encountered in anaerobic digestion. The yield coefficients were determined
based on the bioenergetics described above (assuming ammonia to be the nitrogen
source).
The specific substrate utilization rate can also be calculated based on the
procedures developed by McCarty.43>45>47 However, in this case, the number of
electrons actually transferred must be known when the reaction does not result
in the complete oxidation of the electron donor (such as is the case of fermen-
tations). Only when the actual pathway involved is known can the number of
electrons transferred be accurately estimated. Stoichiometric equations were de-
veloped for the anaerobic conversion of complex substrates (carbohydrates, pro-
tein, fat) to volatile fatty acids (VFA) and the subsequent conversion of VFA
and hydrogen gas to methane. For the conversion of CO2 to CH, using H 2 as the
electron donor, autotrophy was assumed (i.e., CO2 was the only carbon source).
The estimated biokinetic coefficients are reported in Table 3. The overall stoi-
chiometric equations, normalized to 1 mol of electron donor, are presented below.
In each substrate category, the fractions of substrate used for cell synthesis (s)
and for energy production (respiration) (e) are also provided.
a. Carbohydrates (e = 0.504; s = 0.496)

+ 0.595NH4+ + 1.458HCO3- = 0.595C5H7O2N + 0.535CH3COO-
+ 0.190CH3CH2COO-
+ 0.140CH3CH2CH2COO-
+ 2.272CO2 + 2.898H2O
423
0.40
COD)
0.30
o
CO
CO
0.20
3
VIELD 0.10
0.00
O 10 20 30 40
SOLIDS RETENTION TIME (Days)

0.06
.BUTYRATE
B
o
o 0.05
o
CO 0.04
CO -sPROPIONATE
3 0.03 ——.
~~- ^
ACETATE
o
w 0.02
HYDROGEN
0.01
0 10 20 30 40
FIGURE 3. Effect of solids retention time on the observed yield

coefficient. (A) Conversion of carbohydrates, protein, and fat to
VFA. (B) Conversion of butyrate and propionate to acetate, CH4
and CO2 and acetate and H2 to CH4 and C0 2 (yields derived
based on bioenergetics, see text).
b. Protein (e = 0.710; s = 0.290)
C 4 H 6 0N + 1.478H2O + O.lMHCOf = 0.247C5H7O2N + O.3OOCH3COO-

+ 0.280CH3CH2COO-
+ 0.286CH3CH2CH2COO-
+ 0.753NH4+ + 0.298CO2
424
TABLE 3
Biokinetic Coefficients for the Anaerobic Conversion of Various
Substrates (Calculated Based on Bioenergetics)
A
(eeq ed to
Qrnln
energy/eeq V k»
Substrate cell formed) (gVSS/gCOD) (gCOD/gVSS-d) (d)°
Carbohydrates 1.02 0.350 15.9 0.18

Protein 2.45 0.205 11.3 0.43
Fat 17.75 0.038 8.5 3.19
Propionate 18:27 0.037 8.4 3.32
Butyrate 11.13 0.058 8.7 2.02
Acetate 21.03 0.032 8.4 3.86
Hydrogen gas 22.55 0.030 29.2a 1.15
True yield coefficient.

Assuming km = 1 eeq of ed to energy/gVSS-d, except for H2
Assumed/) = 0.01 d" 1 .
Assuming km = 3.5 eeq of ed to energy/gVSS-d.48
c. Fat (e = 0.948; s = 0.052)
C15H3,COO- + 0.235NH4+ + 6.822HCO3~ = 0.235C5H7O2N

+ 7.587CH3COO-
+ 3.319CH4
+ 3.850CO2 + 0.530H2O
d. Butyrate (e = 0.918; s = 0.082)
CH3CH2CH2COO- + 0.082NH4+ + 0.918HCO3" = 0.082C5H7O2N

+ 1.835CH3COO"
+ 0.459CH4
+ 0.377CO2
+ 0.165H2O
e. Propionate (e ~ 0.949; s = 0.051)
CH3CH2COO- + 0.036NH4+ + 0.407H2O = 0.036C5H7O2N

+ 0.949CH3COO~
+ 0.711CH4 + 0.196CO2
+ 0.016HCO3-
425
f. Acetate (e = 0.955; s = 0.045)
CH3COO- + 0.018NH4+ + 0.027CO2 + 0.928H2O = 0.018C5H7O2N

+ 0.955CH4
+ 0.982HCO3-
g. Hydrogen (e = 0.958; s = 0.042)
H2 + 0.256CO2 + O.OO4HCO3- + 0.004NH4+ = 0.004C5H7O2N

+ 0.239CH4 + 0.517H2O
From the data in Table 3, we can conclude that carbohydrates and protein
result in higher yield coefficients than fat and VFA since the former substrates
release more energy per electron equivalent of substrate converted. As a result,
the organisms mediating the anaerobic conversion of carbohydrates and protein

have more energy available which allows them to grow faster. The minimum
retention time for these microorganisms is one order of magnitude lower than the
minimum retention time of the fat- and VFA-degrading microorganisms (see Table
3). This observation agrees with the results of O'Rourke,17 who found that, during
the anaerobic digestion of primary sludge, the protein and carbohydrate-fer-
menting bacteria grew faster, resulting in these substrates being rapidly degraded
to fatty acids in relatively short retention times (less than 2.5 d at 35°C). However,
the fermentation of the produced fatty acids required higher retention times (more
than 5 d at 35°C).
Generally speaking, the biokinetic coefficients estimated by the use of bio-
energetics are in agreement with literature data obtained by experimentation. The
yield coefficients agree quite well with published data. For example, the yield
coefficient for the acidogenic conversion of carbohydrates ranges between 0.14
and 0.17 and the yield coefficient for the aceticlastic methanogenesis ranges
between 0.020 and 0.054 (See Section IV). As mentioned before, in the case of
fermentations, the estimation of the specific substrate utilization rate requires
detailed knowledge of the pathways involved for the estimation of the number
of electrons actually transferred from the electron donor to the electron acceptor.
Due to this limitation, and in view of the assumptions made, the reported values
for the specific substrate utilization rate are less accurate than the estimated yield
coefficients. Nevertheless, the data reported in Table 3 indicate that carbohy-
drates, protein, and hydrogen gas result in higher specific substrate utilization
rates than fat and VFA.
Another important use of stoichiometric equations is the estimation of nutrient
requirements. This is especially useful in the case of industrial wastewaters where
nutrient imbalances are common. Anaerobic bacteria are more fastidious than
their aerobic or facultative counterparts. By use of stoichiometric equations, the
nitrogen requirements can be estimated. The phosphorus requirements are about
15% of the nitrogen requirement.49 The anaerobic bacteria, especially the meth-
anogens, have unique nutrient requirements (e.g., Ni, Co, and other cofactors).
Failure to satisfy the trace nutrient requirements may be responsible for many
unsuccessful anaerobic treatment studies of industrial wastewaters.42
426
IV. INTRINSIC KINETIC RATES
Although the rate-limiting step approach (see above) leads to relatively simple
mathematical descriptions of the anaerobic digestion process, a more universal
and fundamental approach is required for a better understanding of the process,
which can only be satisfied by examining each major step of the anaerobic process
separately. This section deals with the intrinsic kinetic rates of the major steps •
of the anaerobic treatment process as depicted in Figure 1.
A. Hydrolysis of Participate Organic Matter
Organic polymeric materials cannot be utilized by microorganisms unless

they are broken down to soluble compounds (usually mono- or dimers) which
can then pass the cell membrane. Therefore, solubilization is the first step in the
anaerobic degradation of complex polymeric organics. In terms of chemical com-
position, three groups of organics are considered as the major components of
complex organics: carbohydrates, proteins, and lipids. Although kinetic infor-
mation on each chemical component is desirable, literature data are scarce and
in most cases deal only with complex organic matter with no reference made to
the chemical composition of the material. In addition, differences in the conditions
of the various studies (e.g., batch vs. continuous systems) make interstudy com-
parisons very difficult. A summary of the pertinent literature is presented in this
section.
1. Hydrolysis of Carbohydrates
Lignocellulosic materials (composed of cellulose, hemicellulose, and lignin)

are the most abundant natural organic compounds. These materials are usually
subject to anaerobic degradation in natural systems (e.g., swamps), the rumen
of herbivores, and in anaerobic digesters. In the latter case, the origin of the
plant-derived materials is either agricultural (e.g., wheat straw as part of animal
manure, food processing waste) or paper products (e.g., municipal refuse). The
hydrolysis products of cellulose are cellobiose (a dimer) and glucose whereas
hemicellulose hydrolyzes to pentoses, hexoses, and uronic acids.50 Lignin is
highly recalcitrant and, because of its association with cellulose, lignin decom-
position is considered to be the rate-limiting step in the degradation of lignocel-
lulosic materials.
Most of the literature data on hydrolysis of carbohydrates comes from studies
dealing with the hydrolysis of cellulose by pure cultures usually encountered in
the rumen of plant-eating animals (herbivores). Stack and Cotta51 have shown
that cellulose disappearance in batch cultures of Ruminococcus albus strain 7 was
first-order with a rate constant equal to 2.88 d""1 for cultures grown at 37°C on
ball-milled Whatman No. 1 filter paper. Pavlostathis et al.52-53 found that the
427
hydrolysis and fermentation of cellulose (Avicel, type PH-105) by continuous
cultures of R. albus strain 7 followed first-order kinetics and the rate constant
was equal to 1.18 d ~ '. The concentration of soluble reducing sugars was negligible
(<1%) compared with the amount of insoluble cellulose used. These data show
that the rate-limiting step in the fermentation of cellulose by R. albus is the
conversion of insoluble cellulose to soluble substrates which are then rapidly
fermented to products such as acetate, ethanol, formate, H2, and CO2.
Data of batch cellulose fermentation by the wild type of Clostridium ther-
mocellum at 60°C reported by Tailliez et al.54 were used and the first-order model
for cellulose degradation was applied resulting in a rate constant equal to 0.42
± 0.02 d - I (mean ± 8 0 ; ^ = 0.94). The data reported by Lynd et al. ss on the
continuous culture of C. thermocellum grown at 60°C on cellulose (Avicel, type
PH-105) and dilute-acid-pretreated hardwood were used to fit the first-order model.
The calculated rate constant was equal to 0.15 ± 0.01 d" 1 (mean ± SD; r2 =
0.91) and 0.038 ± 0.005 d"1 (mean ± SD; r2 = 0.69) for Avicel and pretreated
wood, respectively. The lower rate constant for the hydrolysis and fermentation
of pretreated hardwood compared to the rate achieved for the Avicel might be
attributed to physical/chemical properties of the substrates (e.g., substrate particle
size, presence of lignin).
A comparative study on the acid-phase anaerobic digestion of cellulose,
soluble starch, and glucose has recently been performed.56 The cellulose degra-
dation data were fitted to the Contois-Chen and Hashimoto model. The degra-
dation data of soluble starch and glucose were fitted to the Monod model. Hy-
drolysis of cellulose was shown to be the rate-limiting step.
Semi-continuous anaerobic digestion for the production of biogas from corn
stover (20 mesh) at 35°C followed the first-order model.57 The reported constant
was 0.045 d" 1 . However, when the nondegradable portion of this substrate (20%)
was taken into account, the calculated rate constant using the reported data was
found equal to 0.076 ± 0.003 d" 1 (mean ± SD; r2 = 0.98). The introduction
of a culture of C. butyricum into the anaerobic digester resulted in an increased
rate constant of 0.053 d" 1 (without accounting for the nondegradable portion of
the substrate). The degradation rate was recalculated, taking into account the
nondegradable portion of the substrate (20%) resulting in a rate constant 0.18 ±
0.04 d" 1 (mean ± SD; r2 = 0.78). These results illustrate two major points: (1)
failure to account for the nondegradable portion of the substrate erroneously leads
to reduced rates; and (2) substrate conversion rates depend on the type of the
prevailing microorganisms which in turn depend on the type of substrate and the
origin of the inoculum. Data on the anaerobic degradation of carbohydrates are
summarized in Table 4.
2. Hydrolysis of Proteins
Proteins are hydrolyzed by extracellular enzymes, called proteases, into poly-

peptides and amino acids. According to Lackey and Hendrickson,62 the general
428
TABLE 4
Apparent First-Order Degradation Rate Contant (fr, d~1) for Various
Carbohydrates
First-order
rate
Temp. constant*
Substrate Process type/culture (d- 1 ) Reí
ro
Cellulose" 30-35 Semi-continuous/mixed 0.05° 58
Cellulose" 28 Batch/mixed 0.17 59
Cellulose 35 Semi-continuous/mixed 0.28-0.52 49
Cellulose 35 Batch/mixed 0.12 60
Cellulose' 37 Balch/Ruminococcus albus 2.88 51
Cellulose' 37 Continuous^, albus 1.18 53
Cellulose 60 Batch/Clostridium thermocellum 0.42 54

Cellulose1 60 Continuous/C. thermocellum 0.15 55
Starch 35 Semi-continuous/mixed 0.20-1.08 49
Straw/pig manure 35 Batch/mixed 0.02 61
Calculated from reported data.

Component of cattle manure.
Assumes 70% ultimate biodegradability.
In sewage sludge.
Ball-milled Whatman No. 1 filter paper.
Avicel, type PH-105.
scheme of enzymatic protein breakdown proceeds through the following steps:

Protein —> Proteoses —> Peptones —> Peptides —> Amino Acids. Proteases are of
two types: the extracellular, known as proteinases, which attack the whole protein,
and the intracellular, known as peptidases, which cut amino acids off the end of
proteins or polypeptides. Apparently, peptones are the largest protein residues
which can pass through the bacterial cell wall. Comparatively few organisms are
capable of excreting proteinases into their environment in any sizable quantities.
In most cases, a readily utilizable nitrogen source is required to enable the or-
ganism to synthesize the necessary extracellular enzymes.
The amino acids produced as a result of protein hydrolysis are further fer-
mented to volatile fatty acids, carbon dioxide, hydrogen gas, ammonium, and
reduced sulfur (i.e., S2~). Hydrolysis and fermentation are usually carried out
by different groups of bacteria, the latter process mediated by sulfate-reducing
and/or H2-producing syntrophic bacteria grown in association with the fermen-
tative bacteria. Solubility, the kind of end group, tertiary structure, and pH have
been found to affect the rate and extent of protein degradation.63
Generally speaking, the hydrolysis of protein under anaerobic conditions is
slower than the hydrolysis rate of carbohydrates.64 Assuming first-order kinetics
for the degradation of proteinaceous matter, based on reported data, the apparent
rate constant was calculated for various substrates and results are shown in Table
429
5. More information on protein hydrolysis is presented later based on studies
dealing with the degradation of complex substrates.
3. Hydrolysis of Lipids
The degradation of lipids in anaerobic environments proceeds through the

initial breakdown of fats by a group of esterases, called lipases, to their constituent
long-chain fatty acids and the galactose and glycerol moieties. Upon complete
hydrolysis, phospholipids yield one equivalent of glycerol, one equivalent of
phosphoric acid, and two equivalents of fatty acids. The long-chain fatty acids
are degraded by ß-oxidation, as is discussed later. The glycerol portion of the
lipids is probably degraded via glyceraldehyde phosphate and pyruvic acid.62 The
lipid hydrolysis products are further fermented to volatile fatty acids, carbon
dioxide, and hydrogen gas.63 Although domestic sewage sludge and other wastes
(e.g., meatpacking and slaughterhouse wastes) are high in lipids, relatively little
is known about anaerobic lipid degradation and most of the available information
is derived from studies dealing with the rumen of herbivoric animals.63 In view
of the lack of literature data on the hydrolysis of lipids, most available information
TABLE 5
Apparent First-Order Degradation Rate Constant (k, d~') for
Various Proteinaceous Substrates
First-order
Temp. rate constant*
Substrate (°C) Process type (d-1) Reí
Albumin 37 Batch 0.57 65

Alanine 37 Batch 0.92 65
Casein 37 Batch 0.35 65
Gelatin 37 Batch 0.60 65
Glycine 35 Semi-continuous 0.44-0.98 49
Glycine 37 Batch 0.19 65
Glygine 35 Batch 0.10 60
Glutamic acid 35 Semi-continuous 1.12 66
Leucine 35 Semi-continuous 0.31-0.63 49
Leucine 37 Batch 0.13 65
Valine 37 Batch 0.18 65
Zein" 35 Batch 0.04 60
Nutrient broth 35 Semi-continuous 0.27-1.3 49
Complex substrate0 37 Continuous 0.10 37
Calculated from reported data.

Maize protein (rate constant calculated based on reported methane pro-
duction data).
Tryptone, beef extract, yeast extract; assuming feed protein carbon 35%
of total carbon.
430
is presented later based on studies dealing with the lipid component of complex
wastes.
4. Hydrolysis of Complex Organic Substrates
Pfeffer67 reported data obtained from laboratory studies dealing with the
digestion of a mixture of primary and waste activated sludge. Based on these
data, Gujer and Zehnder8 calculated first-order decay rates of 0.077 d" 1 at 25°C
and 0.15 d" 1 at 35°C assuming that the degradable fraction of the sludge volatile
solids was 0.80. When the first-order model was used to depict the data obtained
from the fermentation of domestic refuse (municipal solid waste) at a temperature
range of 35 to 60°C, the rate constant varied from 0.052 to 0.99 d" 1 for short
retention times (below 15 d) and from 0.007 to 0.42 d" 1 for the longer retention
times (above 15 d). 21

Foree and McCarty68 studied the decomposition of algae by methane fer-
mentation and sulfate reduction. The solubilization of the biodegradable fraction
of algal biomass was described by first-order decay kinetics. The rate constant
for different species of algae at 20°C had a mean value of 0.22 d" ' and a range
of 0.11 to 0.32 d" 1 .
Eastman and Ferguson18 studied the solubilization of primary sludge and
derived a kinetic model for acid-phase digestion. They concluded that the rate-
limiting step in the conversion of waste solids to fermentation products in the
acid phase of anaerobic digestion is the hydrolysis of particulate matter to soluble
substrates. The soluble organic carbon produced during the acid phase consisted
primarily of volatile acids (85 to 95% of the soluble COD). The lipid fraction
was not degraded during this study (retention time less or equal to 3 d). Loss of
COD to the gas phase could be restricted to <10% of the product COD with
control of solids retention time. The methane produced at low solids retention
times was attributed to CO2 reduction by hydrogen-utilizing bacteria. The hy-
drolysis of the degradable, particulate COD was modeled assuming first-order
kinetics. At 35°C, the hydrolysis rate constant was found to be 0.125 h~' ( =
3.0 d" 1 ). Increasing values of pH improved the solubilization of the particulate
substrate. Based on the influent particulate COD and the particulate COD utilized
as estimated by Eastman and Ferguson,18 the apparent first-order hydrolysis rate
constants were calculated for the nitrogenous, carbohydrate and total COD as a
function of pH with results shown in Table 6.
O'Rourke17 evaluated the methane fermentation kinetics of a complex waste
(municipal raw sewage sludge) by operating semicontinuously fed digesters at
several values of solids retention time and at temperatures of 15, 20, 25, and
35°C. O'Rourke observed that — for any retention time above that causing
washout of fatty-acid utilizing bacteria — the degradable material in the effluent
consisted mostly of long- and short-chain fatty acids. From such observations,
he concluded that fatty-acid utilization was the "rate-limiting" step in the an-
aerobic digestion of primary sewage sludge. It was further shown that hydrolysis
431
TABLE 6
Apparent First-Order Hydrolysis Rate Constant {kh, d~1) for Various
Components of Primary Sludge
Rate constant, kh (d~1) for

pH Nitrogenous COD Carbohydrate COD Total COD
5.14 0.28 0.30 0.11

5.85 0.39 0.41 0.14
6.67 0.69 0.58 0.20
Note: e = 1.5 d; 35°C; kh was calculated assuming all components are totally biodegradable.
Based on data reported by Eastman and Ferguson.18

of the triglycérides, which accounted for 66% of the lipid COD, was essentially
complete at solids retention times less than those required for effective methane
fermentation of the hydrolysis products (i.e., the long-chain fatty acids). Thus,
for all practical purposes, O'Rourke considered lipids and long-chain fatty acids
to be indistinguishable. Based on the data reported by O'Rourke,17 the apparent
first-order degradation rate constant for protein, cellulose, lipids, and total COD
was calculated (considering only the biodegradable fractions of the sludge con-
stituents as reported by O'Rourke). Generally speaking, for a given retention
time above washout, the degradation rate increases with increasing incubation
temperature. For example, at 35°C and a range of solids retention times of 7.5
to 30 d, the range of the calculated apparent first-order constant is as follows 0.2
to 0.8 d" 1 for protein; 2.4 to 41 d" 1 for cellulose; 0.3 to 0.7 d" 1 for lipids, and
0.4 to 1.2 d~' for total degradable COD.
Ghosh38 presented a kinetic model of the acid-phase fermentation of a simple
substrate (glucose) and a complex substrate (activated sludge). The development
of this model was based on microbial growth following the Monod equation and
product formation rate and efficiency as formulated by Pirt.69 Acid-forming bac-
teria grown on glucose exhibited a maximum specific growth rate one order of
magnitude higher and a half-velocity constant three orders of magnitude lower
than those obtained with the sludge substrate. The acetate formation efficiency
was much higher with glucose than with sludge. Assuming that the degradation
of both substrates follow first-order kinetics and based on the data reported by
Ghosh,38 the rate constant values vary from 15.7 to 80.0 and 0.007 to 0.025 h~'
for glucose and sludge, respectively. Ghosh concluded that the rate-limiting step
in sludge acid-phase digestion was hydrolysis.
Gossett and Belser,39 recognizing the interrelationship of both the aerobic
and the anaerobic processes dealing with the digestion of waste activated sludge,
undertook a study with the goal of predicting the dependence of anaerobic digester
performance upon the activated sludge solids retention time. Two models were
proposed: (1) a model relating the ultimate anaerobic biodegradability of activated
432
sludge to the solids retention time and other parameters of the sludge-generating
process; and (2) a kinetic model for the anaerobic digestion of waste activated
sludge. Assuming that the biodegradable fraction of waste activated sludge con-
sists almost exclusively of the biodegradable portion of viable, activated sludge
organisms, the following equation was developed for the prediction of the acti-
vated sludge's ultimate biodegradability when the influent to the activated sludge
process does not contain any volatile suspended solids:39
where D = ultimate biodegradability of activated sludge (fraction); fd = net

biodegradable fraction of active biomass; b = activated sludge microorganism
decay coefficient (d" 1 ); and 8C = solids retention time in the activated sludge
process (d).
When Gossett and Belser39 used a model, largely based on the O'Rourke
model,17 to explain the performance of anaerobic digesters fed with waste acti-
vated sludge, the model consistently predicted higher digester performance. These
researchers hypothesized that anaerobic digestion of activated sludge may require
a preliminary conversion of the potentially degradable portion of activated sludge
organisms to available substrate via some combination of autolysis, endogenous
fermentative decay, or hydrolysis. The conversion mechanisms, although not
delineated, were lumped together into a so-called "availability rate" which was
assumed to be first-order. A specific availability rate coefficient of 0.22 d~ ' was
estimated from data fitting.
In a subsequent study,2(M<)>41 a model was developed for the anaerobic diges-
tion of biological solids by incorporating preliminary conversion mechanisms.
According to this model, viable activated sludge cells first undergo death/lysis
(processes presumed to be essentially concurrent), followed by hydrolysis to
become available substrate for the anaerobic digester microflora. The death/lysis
of activated sludge cells in the digesters was found to follow first-order kinetics
with a rate coefficient equal to 2 d" 1 . Upon death, the cell membrane ruptures
(i.e., lysis occurs) and part of the intracellular, soluble, degradable matter is
immediately released. This fraction was estimated as equal to 30% of the total
cell degradable matter. The resulting particulate, degradable matter is subject to
extracellular hydrolysis, primarily induced by the active digester microorganisms.
The hydrolysis of the dead particulate, degradable activated sludge cell matter in
the digester followed first-order kinetics with a rate coefficient ranging between
0.14 and 0.16 d~ ! . Particulate protein accounted for >80% of the degradable
COD in the digester effluents. Carbohydrate concentrations were low, and soluble
lipids and volatile acid concentrations were very low (see Figure 4). These results
indicate that hydrolysis of particulate protein is the major, rate-limiting step in
the anaerobic digestion of biological solids.
Dinopoulou et al.70 investigated the effect of operational parameters, such as
433
10
-1 O TOTAL COD
3 8 O PROTEIN
o o CARBOHYDRATES
O
6 A LIPIDS
o
LU
_l
m
<
CE 2
O
UJ
Û
O
8 10
FIGURE 4. Effluent composition from continuous-flow digesters fed biological sludge.

(Data from Reference 20.)
hydraulic retention time, organic loading rate, influent substrate concentration,

pH, and temperature, on the performance of a CSTR-type acidogenic digester
fed with a complex substrate (beef extract). The main fermentation products were
acetic and propionic acids. The degree of acidification increased with retention
time and decreased with organic loading. Under a relatively constant organic
loading rate (8.6 to 9.4 g COD/L-day) at 35°C, the acidification data as reported
by these researchers were used to estimate an acidification rate constant assuming
first-order kinetics. The rate constant varied from 0.039 to 0.133 h" 1 (mean =
0.079 h->; SD = 0.037 h" 1 ; n = 5).
Gujer and Zehnder8 have compiled literature data and calculated the apparent
hydrolysis rate constants for complex biopolymers under anaerobic conditions.
The apparent hydrolysis rate constants for each type of complex substrate varied
as follows: lipids, 0.08 to 1.7 d - 1 ; proteins, 0.02 to 0.03 d" 1 ; cellulose, 0.04 to
0.13 d" 1 ; and hemicellulose, 0.54 d - 1 . '
B. Fermentation and Anaerobic Oxidation of Hydrolysis Products
1. Fermentation of Soluble Carbohydrates
Most of the information available on the fermentation of soluble carbohydrates
434
(i.e., mono- or disaccharides) comes from studies dealing with fermentation
patterns of rumen bacteria. The major products of soluble carbohydrate fermen-
tation by anaerobic bacteria are ethanol, acetate, H2, and CO2 in the absence of
methanogenic bacteria.71-72 However, when H2-utilizing bacteria are present, a
reduction in ethanol and an increase in acetate production are observed. The shift
in the fermentation products is explained by the theory of interspecies hydrogen
transfer which efficiently reduces the hydrogen concentration and raises the redox
potential of the H + /H 2 couple.11-72 When hydrogen is effectively removed, the
anaerobic, fermentative bacteria do not produce electron sink compounds (e.g.,
ethanol) but rather produce H2 from NADH which leads to an increase of the
produced ATP energy.
Numerous studies have been performed dealing with the fermentation of
soluble carbohydrates. Pertinent kinetic data are summarized in Table 7. A com-
mon characteristic of these studies is the almost complete and fast fermentation
of the carbohydrate substrates. For example, Weimer and Zeikus77 found that C.
thermocellum grew faster on cellobiose than on cellulose. The minimum doubling
time of C. thermocellum at 60°C was 2.1 h on cellobiose as opposed to 11 h on
cellulose. Russell and Baldwin78 evaluated the substrate affinity of several rumen
bacteria grown on soluble carbohydrates, such as cellobiose, glucose, maltose,
sucrose, and xylose, in continuous cultures grown at 39°C. The half-velocity
constant (Ks) varied from 0.004 to 11.76 mM whereas the maximum specific
growth rate (|xmax) varied from 0.50 to 20 h" 1 . The observed wide variation in
substrate affinities by the same bacterial species for different substrates and among
species for the same substrate was thought to be responsible for the bacterial
competition in the rumen where the concentration of soluble carbohydrates is
usually low.
2. Fermentation ofAmino Acids
The fermentation of amino acids is a complex process involving redox re-

actions. The major electron acceptors in the reductive reactions used by anaerobic
bacteria are amino acids, keto acids, unsaturated acids, and protons. Short-chain
fatty acids, succinate, aminovalerate, and hydrogen gas are the major end products
from the fermentation of amino acids.63
Oxidative degradation (dehydrogenation) of amino acids in digesting sludge
has been observed where the methanogenic bacteria acted as H2 acceptors. Redox
reactions between suitable amino acids according to the Stickland reaction were
also observed when methanogenesis was inhibited by the addition of chloroform.
These results suggest that amino acids can be degraded by coupled oxidation-
reduction reactions independently from the activity of methanogens as H2 accep-
tors.65 Studies with pure cultures of a ruminai peptostreptococcus grown on
various amino acids produced large amounts of branched chains volatile fatty
acids (e.g., isovalerate, isocaproate when grown on leucine). Valine and isoleu-
cine competitively inhibited leucine transport.79
435
TABLE 7
Summary of Kinetic Data for the Fermentation of Carbohydrates (Acid-Phase Digestion) by Mixed
Cultures
Temp. k Um.» r
Substrate Process (°C) (mg COD/mg VSS-d) g COD/L) (h"1) (mg VSS/mg COD) Ref.
Glucose Batch 35 427 0.3 0.15 73

Glucose Continuous 36.5 22.5 1.25 0.17 6.1 74
Glucose Continuous 37 527 0.323 — 75
Glucose Continuous 37 370 0.30 0.14 76
Cellulose Continuous 35 1.33 8.0a - 56
Glucose Continuous 35 70.6 75.3 — — 56
Starch Continuous 35 40.0 630 56
• K (g COD/g VSS) according to the Chen and Hashimoto model.35

O)
Results on the anaerobic degradation of amino acids produced by the hydro-
lysis of complex proteinaceous wastes indicate very low remaining soluble ni-
trogenous organic matter. For example, the residual soluble nitrogenous material
in a CSTR digester fed with primary sewage sludge and operated at a retention
time of 3 d was 290 mg/L as COD (i.e., 4% of the total degradable COD
utilized).18 Similarly, the steady-state total soluble effluent COD of a digester
fed with a biological sludge and operated at 6.5 d was 150 mg/L compared to a
total effluent proteinaceous COD of ca. 12 g/L (i.e., less than 2%).41 These data
indicate that the fermentation of amino acids produced during the anaerobic
hydrolysis of proteins is fast and that the rate-limiting step in the anaerobic
degradation of proteins is hydrolysis. However, the chemical composition, origin,
and various physicochemical treatments of the primary feedstock may have a
pronounced effect on the rate and extent of amino acid fermentation. For example,
studies with pure anaerobic bacteria grown on acid hydrolysate or enzymatic
hydrolysate of casein resulted in different rates of fermentation and different

fermentation patterns.79-80
3. Anaerobic Oxidation of Long-Chain Fatty Acids
Domestic sewage sludge usually has a high content of lipid and free fatty
acids. The free fatty acids or those produced by the hydrolysis of lipids are subject
to anaerobic oxidation by anaerobic bacteria commonly found in anaerobic en-
vironments. For the degradation of long-chain fatty acids, we have adopted the
term "anaerobic oxidation" as defined by Gujer and Zehnder:8 "the microbial
process in which molecular hydrogen is the main sink for electrons." The oxi-
dation of the electron carriers (e.g., nicotinamide adenine dinucleotide phosphate
(NAD(P)H] and/or ferredoxin) is more subject to inhibition by elevated H2 partial
pressures than the dehydrogenation of pyruvate which takes place during the
fermentation of amino acids and carbohydrates, as discussed earlier.8-81 The major
short-chain fatty acids produced as a result of fermentation of long-chain fatty
acids are acetate or acetate and propionate, depending on whether the long-chain
fatty acids are even- or odd-carbon acids, respectively.82
Medium and long length even-carbon fatty acids were degraded in an an-
aerobic digester by ß-oxidation.83 The major intermediate in the degradation of
octanoic (C-8) and palmitic (C-16) acids was acetic acid. The acetic acid-derived
methane was 72.8 and 68.4% from the degradation of octanoic and palmitic acid,
respectively. Based on the data reported by Jeris and McCarty83 and assuming
first-order kinetics for the overall conversion of these fatty acids, the estimated
rate constant ranged from 0.64 to 0.82 and 0.51 to 0.55 d~' for octanoic and
palmitic acids, respectively, indicating a negative correlation between the deg-
radation rate and the chain length.
During the anaerobic digestion of primary sludge, the degradation rate of the
principal long-chain fatty acids was similar to the degradation rate of acetic and
propionic acids which were the predominant volatile fatty acids.17 O'Rourke
437
concluded that the rate-limiting step in the anaerobic digestion of primary sludge
was the degradation (i.e., oxidation) of the long-chain and volatile fatty acids.
A summary of the kinetic data on the anaerobic oxidation of long-chain fatty
acids as determined by O'Rourke17 is presented in Table 8. Because it was
experimentally impossible to determine directly specific utilization rates for the
long-chain fatty acids, O'Rourke used the yield coefficient Y = 0.04 g VSS/g
COD and a microorganism decay coefficient, b = 0.015 d" 1 as previously
estimated by Lawrence.85 The justification for this procedure is that both long-
and short-chain fatty acids provide the same energy theoretically available for.,
microbial growth per unit of substrate COD fermented to methane.85
Novak and Carlson84 developed enrichment cultures from municipal sewage
sludge by feeding single, long-chain fatty acids as the sole source of organic
carbon. Kinetic data are also reported in Table 8. A decrease in the degradation
rate was observed with an increasing chain length of the saturated fatty acids and
with a decreasing degree of saturation of the unsaturated fatty acids.
Chynoweth and Mah86 have reported that palmitate degradation was 11 times
faster than the degradation of protein hydrolysate and more than 80 times faster
than glucose in digesting sludge without prior acclimatization. The predominant
product of palmitate oxidation was acetate, and smaller amounts of formate,
butyrate, and propionate were also produced. When methanogenesis was inhibited
by the addition of chloroform, palmitate degradation led to the production of
butyrate primarily and propionate, acetate, and formate secondarily, indicating
that secondary reactions involving acetate and/or propionate were used to remove
hydrogen produced but not utilized by methanogens. The aforementioned re-
searchers pointed out that as long as the digester retention time is longer than
the washout retention time for the microbial population responsible for the oxi-
dation of long-chain fatty acids, lipid fermentation rates can be faster than the
degradation rates of protein and carbohydrates. They also stressed the shortcom-
ings of enrichment cultures fed with synthetic substrates which in turn lead to
the selection of a microbial population different from that in real-life systems.
4. Anaerobic Oxidation of Short-Chain Fatty Acids
Several investigators have reported on the anaerobic oxidation of short-chain

fatty acids (e.g., propionate, butyrate). The main products of the anaerobic ox-
idation of short-chain fatty acids are acetate and hydrogen gas.82-87 These reactions
are usually termed acetogenesis since acetate is the major carbon product. For
the successful degradation of the short-chain fatty acids, efficient removal of the
produced hydrogen gas is a must. As a result, most of the studies dealing with
the anaerobic oxidation of short-chain fatty acids have been carried out as syn-
trophic associations of two or more species.88
Lawrence and McCarty89 operated a series of laboratory digesters with en-
richment cultures developed from municipal wastewater sludge for the evaluation
of the degradation kinetics of short-chain fatty acids. Single volatile acids were
438
8
Kinetic Data for the Anaerobic Oxidation of Long-Chain Fatty Acids
If Y
Process Temp. (mg COD/ K. (mg VSS/
Substrate type (°C) mg VSS-d) (mg COD/L) (d- 1 ) mg COD) b (d-i) Ref
Long-chain fatty Semi-continuous 20 3.85 4620 0.139 0.04" 0.015b 17

acids* 25 4.65 3720 0.171 0.04 0.015
35 6.67 2000 0.252 0.04 0.015
Saturated long- Continuous 37 84
chain fatty acids
Myristic (C-14) 0.95 105 0.105 0.11° 0.01°
Palmitic (C-16) 1.00 143 0.110 0.11 0.01
Stearic (C-18) 0.77 417 0.085 0.11 0.01
Unsaturated long- Continuous 37 84
ES- chain fatty acids
Olelc(C-18) 4.00 3180 0.44 0.11 0.01
Linoleic (C-18) 5.00 1816 0.55 0.11 0.01
Hydrolysis products of primary sludge lipids.

Assumed values based on data reported by Lawrence65 (see text).
Average values; experimental values were reported as Y = 0.10-0.12 mg VSS/mg COD and b = 0-0.02 d" 1
used as the sole organic carbon source and the digesters were fed continuously.
Their kinetic data for the oxidation of propionic and butyric acids are reported
in Table 9.
Continuous-flow anaerobic digesters, developed by the enrichment of sludge
obtained from a mesophilic (35°C) sewage sludge digester, were fed a mixture
of acetic, propionic, and butyric acids on a COD basis ratio of 2:1:1,92 The kinetic
data obtained in this study are reported in Table 9. At high feed substrate con-
centrations (e.g., 70 g COD/L at a hydraulic retention time of 4.43 d), meth-
anogenesis proceeded without any problems. The specific substrate removal rate
of these digesters fed with a mixture of volatile acids is much higher than pre-
viously reported rates when single volatile acids were fed individually.92
When digesting sludge from a full-scale municipal digester (33°C, 40-d re-
tention time) was tested for its propionate degradation rate by spiking propionate
in a lab-scale digester, the rate increased from 3.4 mg propionate-COD/L-h at

steady-state to 21.3 mg propionate-COD/L-h at enzyme saturation conditions.4
The estimated half-saturation constant varied from 5 to 21 mg COD/L. These
results indicate that in the full-scale digester the propionate-degrading system was
saturated only 10 to 15%.
Numerous studies dealing with the anaerobic oxidation of short-chain fatty
acids have been carried out and a summary of pertinent kinetic data is provided
in Table 9.
C. Methanogenesis
1. Aceticlastic Methanogenesis (or Acetate Decarboxylation)
Acetate is the most important substrate for methanogens. In sewage sludge

digesters, about 65 to 70% of the methane produced is via reduction of the acetate
methyl group.83-94 A large number of studies have dealt with acetate conversion
to methane. A summary of the kinetic data for acetate utilization is reported in
Table 10.
Continuous anaerobic digestion experiments with a pure culture of Methan-
osarcina barkeri (DSM 804) grown on acetic acid at 37°C and at a constant pH
= 6.3 were carried out by Wandrey and Aivasidis" and kinetic data are reported
in Table 10. Under the conditions of these experiments, about 97% of the acetic
acid carbon was converted to gas and the rest was incorporated into biomass.
Continuous cultivation of four species of Methanosarcina in a pH auxostat (pH
= 6.8 ± 0.2) using acetic acid as the major carbon and energy source led to
cell yields ranging from 0.019 to 0.042 mg dry wt/mg acetate-COD.102 When
digesting sludge from a full-scale municipal digester (33°C, 40-d retention time)
was tested for its acetate degradation rate by spiking acetate in a lab-scale digester,
the rate increased from 17.3 mg acetate-COD/L-h at steady-state to 26.2 mg
acetate-COD/L-h at enzyme saturation conditions.4 The estimated half-saturation
constant varied from 13 to 29 mg/L (COD basis). These results indicate that in
440
TABLE 9
Kinetic Data for the Anaerobic Oxidation of Short-Chain Fatty Acids (Except Acetate) by Continuous-Flow,
Mixed Cultures
Temp. (mg COD/mg K. Um« Y b

Substrate (°C) VSS-d) (mg COD/L) (d-1) (mg VSS/mg COD) Re1
Propionic 25 7.8 1145 0.358 0.051 0.040 89

Propionic 33 6.2 246 0.155 0.025 — 8
Propionic 35 7.7 60 0.313 0.042 0.010 89
Propionic 35* — 17 0.13 — — 90
Propionic 35" — 500 1.20 — — 90
Butyric 35 8.1 13 0.354 0.047 0.027 89
Butyric0 60 — 12 0.77 — — 91
Mixed acids" 35 17.1 166 0.414 0.030 0.099 92
Butyric 37 — 298 0.86 — — 93
Retention time, 14.5 d.

Retention time, 8.2 d.
Triculture.
Acetic.-propionic.butyric (2:1:1 as COD).
Degradation product of glucose.
TABLE 10
Summary of Kinetic Data for the Aceticlastic Methanogenesis
Temp.
K. Y b
Process type* (°C) (mg COD/mg VSS-d) (mg COD/L) (d-1) (mg VSS/mg COD) (d-') Ref
C/mixed culture 25 5.0 930 0.250 0.050 0.011 89
C/mixed culture 30 5.1
C/mixed culture" 35 356 0.275 0.054 0.037 89
8.7 165 0.357 0.041
C/triculture 60 0.015 89
26 0.28 — 91
B/mixed culture 20 2.6
B/mixed culture 30 — — 0.05 95
2.6-5.1
B/mixed culture 35 — 0.02 95
2.6-5.1 — 0.08-0.09 0.02
B/pure culture 50 95
320 1.4 96
B/pure culture 36
320 0.5-0.7 0.03-0.04 97
C/acetate 35
8.5 185 0.34 0.04 0.036
enrichment 98
CM barken 37 8.6° 257 0.206 0.024 0.004 99
B/Methanobacterium 30 26.0
sp. 11 0.26 0.01 100
C/M. soehngenii 37 30 0.11 0.023 — 101
C/mixed culture
ec = 4.5 d 35 11.6 421 —
eo = 6.5 d 35 56
6.6 43 — —
6C = 9.6 d 35 56
4.4 15 — — 56
" C = continuous; B = batch.
" Mean values of five sets of data.
e
K =
the full-scale digester the acetate-degrading system was saturated by approxi-
mately 50%.
2. Methanogenesis from Hydrogen and Carbon Dioxide
With few exceptions, most methanogenic bacteria use H2 and CO2 for growth.103
The efficient removal of H2 produced during the fermentation of carbohydrates
and proteins and the anaerobic oxidation of fatty acids by methanogens allow the
aforementioned reactions to proceed under natural, physiological conditions. Al-
though about one third of the methane produced in a municipal digester comes
from the reduction of CO2 by H2, the interspecies H2 transfer and utilization is
far more important since it regulates the rate of H2-producing reactions by con-
trolling the partial pressure of hydrogen. Recognizing the regulatory role of H2
in anaerobic environments, numerous studies have been undertaken for the de-
lineation of the kinetics of H2 utilization by methanogenic bacteria. A number of
these studies are summarized herein.
Zehnder and Wuhrmann104 isolated a methanogenic bacterium which uses H2
and CO2 as the sole energy and carbon source. The originally called Methano-
bacterium strain AZ (now called Methanobrevibacter arboriphilus) has an opti-
mum temperature between 33 and 40°C and an optimum pH of 7.0. Kinetic data
for this bacterium grown in the supernatant of digested sewage sludge at pH 7
and at 33°C are reported in Table 11.
Kaspar and Wuhrmann4 tested digesting sludge from a full-scale municipal
digester (33°C, 40-d retention time) for the H2 utilization rate by spiking H2 in
a lab-scale digester. The rate of H2 utilization increased from an average 13.6
mg COD/L-h at steady-state to 1650 mg COD/L-h at enzyme saturation condi-
tions. However, the highest H 2 removal observed (at 0.7 arm H2 partial pressure
and high degree of mixing) was only 325 mg COD/L-h, indicating mass transfer
limitations. The estimated half-saturation constant (Kt) was 0.105 atm. These
results indicate that in the full-scale digester the H2-utilizing system was saturated
by < 1 % .
Hydrogen utilization kinetics have recently been studied in continuous cultures
with a cellulolytic, fermentative bacterium (/?. albus) and an H2/CO2 utilizing
methanogen (Ai. smithif).105 The growth yield of M. smithii was estimated as
YCH4 = 2.8 g/mol. Kinetic constants of hydrogen utilization by the coculture are
summarized in Table 11. The final products of the coculture were primarily
acetate, CH4, and CO2 and low levels of ethanol and H2. The coculture produced
more H2 (used for the reduction of CO2 to CH4) and acetate than a monoculture
of R. albus (see Figure 5). These differences in fermentation products could be
accounted for by the lower production of ethanol which conforms to the theory
of interspecies H 2 transfer. Since the hydrogen was a product of R. albus, gas-
to-liquid mass transfer limitations were avoided which led to a low K, value.
Most of the high Ks values for hydrogen uptake by methanogens (see Table 11)
443
TABLE 11
Summary of Kinetic Data for Methanogenesis from Hydrogen and Carbon Dioxide
Temp. k Hrn., Y b
Culture (°C) (mg COD/mg VSS-d) (mg COD/L) (d- 1 ) (mg VSS/mg COD) (d- 1 ) Ref.
Methanobrevibacter 33 0.6 1.4 0.04 — 8

arboriphilus
M. smithii* 37 90 0.018 4.02 0.045 0.088 105
M. smith iib 37 — — 4.07 — — 106
Rumen bacteria 37 2-8° 0.016 — — 107
Rumen fluid 39 213-453" 0.067-0.145 — — 108
Digester sludge 30 11-69" 0.07-0.109 — — — 108
Lake sediment 9 2-7.8" 0.09-0.137 — — — 108
Methanobacteríum 60 50-54 0.093-0.136 — 0.13 — 91
thermoautotrophicum •
Methanobrevibacter 35 46.1 0.105 — — — 109
arboriphilus*
Methanospirillum hungatei 37 1.92' 0.093-0.117 0.05 0.017-0.025' — 110
JF-1"
Mixed culture0 35 16.5" 4.8 x 10- 5 — 111
In coculture with the cellulolytic bacterium Ruminococcus albus.

Batch grown on H2:CO2 gas mixture.
Expressed as mg COD/g rumen liquid-day.
Expressed as mg COD/L-h.
In a butyrate-degrading triculture.
Assuming a protein content of 60% of dry weight.
Grown on propionate.
•o
CO 320
CO MONOCULTURE (R. albus)

o
o
"I 240 COCULTURE (R. albus & M smithii)
co
75
o 160
o
co
•2
"5
•§ 80
CO
O
O
g
CL
° ACETATE ETHANOL FORMATE CO 2
FIGURE 5. Fermentation products of a monoculture and a coculture showing the effect

of H2 removal by the methanogenic bacteria on the fermentation pattern. (Data from Ref-
erence 105.)
are apparent values reflecting mass transfer limitations for externally supplied
substrate (i.e., exogenous hydrogen gas).
A review of the kinetics and energetics of hydrogen gas in anaerobic waste-
water treatment has recently been published by Harper and Pohland.112 Kinetic
values for methane production using H2 and CO2 as the electron donor and electron
acceptor, respectively, are summarized in Table 11.
3. Substrate Thresholds in Methanogenesis
Recently there have been several reports in the microbiological literature on

the existence of thresholds for substrate uptake by methanogens. A threshold is
considered to be the concentration of substrate below which the substrate con-
sumption stops. Lovley113 proposed that methanogens are outcompeted by sulfate
reducers in sediments by maintaining the hydrogen partial pressure below the
threshold for methanogenesis. Cord-Ruwish et al.114 have investigated the nature
of the substrate threshold in different hydrogenotrophic bacteria and concluded
that the threshold depends on the redox potential of the terminal acceptor (e.g.,
nitrate, sulfate, carbon dioxide). Electron acceptors with increasing redox poten-
tial resulted in decreasing hydrogen thresholds. It has been reported that hydrogen
consumption during methanogenesis by mixed cultures111 and defined cocultures115
445
did not follow Michaelis-Menten kinetics. Table 12 summarizes information on
hydrogen thresholds for methanogens.
Similarly, thresholds have also been reported for acetotrophic methanogens
and for hydrogenotrophic methanogens that use acetate as carbon source. Wes-
termann et al.117 proposed that acetate thresholds in acetotrophic methanogens
are responsible for the predominance oîMethanotrix species over Methanosarcina
species at low acetate concentrations. Min and Zinder118 first reported that acetate
utilization by two thermophilic acetate decarboxylating methanogens did not fol-
low Michaelis-Menten kinetics. Similar results have been reported by Jetten et
al.119 Fukuzaki et al.120 have recently proposed that the substrate for acetotrophic
methanogens is the undissociated fraction of acetic acid and they have expressed
the threshold concentrations as such. Table 13 summarizes the information on
acetate thresholds by acetotrophic methanogens.
The nature of the thresholds has not yet been elucidated. Thresholds seem
to be related to the thermodynamic conditions of the reaction and also to the
bacterial species and the physiological conditions of the microorganisms. Min
and Zinder considered that the thresholds may represent the concentration at which
the microorganism can no longer obtain energy to support the utilization of the
substrate.118
Perhaps the most important consequence of the substrate threshold phenom-
enon, from the perspective of this review, is the failure of the Michaelis-Menten
kinetics to mathematically represent substrate consumption by methanogens. Mi-
chaelis-Menten kinetics overestimate substrate consumption at low concentrations
and underestimate it at concentrations higher than the half-saturation constant.
TABLE 12
Summary of Threshold Values for Hydrogen Uptake during
Methanogenesis
Temperature Threshold
Culture (°C) (ppm)- Ref.
Methanobacterium formicicum 39 65 113

M. bryantii 39 69 113
Methanospirillum hungatei 39 95 113
M. hungatei 28-34 30 114
Methanobrevibacter smithii 28-34 100 114
M. arboriphilus 28-34 90 114
Methanobacterium formicicum 28-34 28 114
Methanococcus vannielii 28-34 75 114
Syntrophomonas wolfei/Methanobacterium 37 45 116
fonvidcum
Digester mixed culture 35 3 111
Defined coculture 37 36 115
• Gaseous phase concentration (v/v).
446
TABLE 13
Summary of Threshold Values for Acetate Uptake during
Methanogenesis
Temperature Threshold
Culture (°C) (mg/L) Ref.
Methanosarcina barken 37 70.8 117

M. maze} 37 23.8 117
Methanothrix sp. 37 4.1 117
Methanosarcina sp. 58 60-90 118
Methanothrix sp. 58 0.72-1.26 118
Methanothrix soehngenii 37 0.42 119
Methanosarcina barkerii 37 14.6 119
Methanobacterium NR" 20.7 119
thermoautotrophicum
Methanospirillum hungateii NR 33.6 119
Methanobrevibacter arboriphilus NR 91.8 119
Methanosarcina barken 37 15.6-130" 120
Granular sludge 37 0.25 119
NR, not reported.

Values are pH dependent.
Similar results have been reported by Min and Zinder118 and Jetter et al.119 This
quick saturation near the region of the threshold value has been considered as
evidence that ATP generation limits substrate activation or substrate transport in
this range.119 Giraldo-Gomez et al. have proposed a simple modification of the
Michaelis-Menten kinetics that accounts for the threshold value and provides a
better description of the experimental data. ' ' ' The mathematical form is as follows:
V
y _ mn(S - Tr)
K + (S- Tr)
where V is the substrate utilization rate, 5 is the substrate concentration, Km is

the half-saturation constant, and Tr is the threshold substrate concentration. The
above equation is valid for S > Tr. When 5 =s Tr, then V = 0.
The threshold phenomenon has an important implication for the minimum
effluent oxygen demand that can be obtained by anaerobic digesters. This is
especially true for acetate. Depending on the predominant acetotrophic methan-
ogen (e.g., Methanosarcina or Methanotrix), significant acetate-COD concen-
trations can remain untreated in the effluent. For Methanosarcina, a value between
15 and 130 mg/L is expected based on threshold values reported in the literature
(see Table 13).
447
D. Overall Kinetics and Summary of Intrinsic Kinetic Rates
A large number of studies, especially those dealing with undefined, complex

substrates, have yielded values of kinetic parameters without reference to the
individual reactions discussed previously but have assumed that digestion is a
two-step process: acidogenesis and methanogenesis. A summary of kinetic data
from these studies has been compiled by Henze and Harremoes.121 Based on an
extensive literature review, these researchers proposed a set of kinetic values
thought to be representative of the acid-phase and the methane-phase of anaerobic
digestion (see Table 14).
The anaerobic degradation of particulate substrates requires hydrolysis (or
liquefaction) to render these substrates available to the anaerobic microflora. The
hydrolysis step is usually assumed to follow first-order kinetics. In most cases,
the Monod model (or variations thereof) has been found to be inadequate in
describing the heterogeneous reactions taking place during hydrolysis of complex,
particulate substrates. Based on the literature data presented in this paper, when-
ever the kinetics of the hydrolysis step were studied, they were usually found to
be the limiting step in the overall conversion of complex substrates to methane.
With the exception of the hydrolysis step, all other subprocesses of anaerobic
treatment have been successfully modeled by following Monod kinetics. A sum-
mary of reported values for the kinetic constants pertaining to each subprocess
is given in Table 15. The tremendous variation in the reported values does not
allow the derivation of mean, representative kinetic values for each subprocess.
Interstudy comparisons are almost impossible due to the variability in mode of
operation (e.g., batch vs. continuous), as well as environmental and operational
conditions (e.g., pH, organic loading). In addition, most of the oldest studies
have undoubtedly suffered from a lack of advanced instrumentation to enable the
detection and accurate quantification of low concentrations of analytes. This is
particularly true in the case of hydrogen gas. Advanced instrumentation capable
of detecting very low concentrations of intermediates encountered in anaerobic
treatment along with an improvement of the experimental methodology are re-
quired for the more accurate estimation of the pertinent biokinetic constants.
TABLE 14
Representative Values of Kinetic Constants for Anaerobic Digestion
at 35°C
k Y K.
Process (mg COD/mg VSS-d) (mg VSS/mg COD) (mg COD/L) (d-1)
Acidogenesis 13 0.15 200 2.0

Methanogenesis 13 0.03 50 0.4
Overall 2 0.18 0.4
Data of Henze and Harremoes.121
448
TABLE 15
Summary of Values of Kinetic Constants for Various Substrates Utilized in Mesophilic Anaerobic
Treatment Processes
k Y
(g VSS/
(g COD/ K. b
Substrate Process g VSS-d) (mg COD/L) g COD)
Carbohydrates Acidogenesis 1.33-70.6 22.5-630 7.2-30 0.14-0.17 6.1

Long-chain fatty acids Anaerobic 0.77-6.67 105-3180 0.085-0.55 0.04-0.11 0.01-0.015
oxidation
Short-chain fatty acids« Anaerobic 6.2-17.1 12-500 0.13-1.20 0.025-0.047 0.01-0.027
oxidation
Acetate Aceticlastic 2.6-11.6 11-421 0.08-0.7 0.01-0.054 0.004-0.037
methanogenesis
Hydrogen/carbon Methanogenesis 1.92-90 4.8 x 0.05-4.07 0.017-0.045 0.088
dioxide 10-'-0.60
Except acetate.
V. EFFECT OF TEMPERATURE ON KINETICS
As in all biological processes, anaerobic processes are affected by temper-

ature. Generally speaking, the higher the temperature, the higher the microbial
activity until an optimum temperature is reached. A further increase of the tem-
perature beyond its optimum value results in a precipitous decrease in activity.
The most widely used equation for microbial activity-temperature correlations is
the Arrhenius equation:
(24)
where V is the process rate; A is the frequency factor (same units as V); Ea is the
apparent activation energy (kcal mol" 1 ); R 1S the gas constant ( = 0.001987 kcal
mol" 1 K" 1 ); and T is the absolute temperature (K).

A commonly used parameter for the quantification of the temperature effect
on the microbial activity is the temperature coefficient (Ql0) for a 10°C temperature
increase defined as the ratio of a microbial process activity at two temperatures,
T2 and Tu where T2 = 7, + 10. By use of Equation 24 and the above definition,
the following equation is obtained:
(25)
Hinshelwood122 proposed the following model for the effect of temperature

on the net microbial activity by recognizing that there are two opposing processes
(a synthetic and a degradative process):
(26)
where Ea2 > Eal (subscripts 1 and 2 refer to synthesis and degradative processes,
respectively). Equation 26 shows that at low temperatures (i.e., T < Toptimum) the
degradative process is insignificant and the net result is an increase in microbial
activity. However, when the temperature reaches its optimum value, the effect
of the degradative processs is much higher than the synthetic process leading to
a sharp decrease in the net microbial activity. The above-described behavior can
be explained by enzymatic activation and inactivation due to a temperature in-
crease. In addition to the denaturation of proteins at relatively high temperatures,
lysis of cells has been observed to increase sharply with increasing temperature,
especially when the substrate is exhausted.123
Although microbial methane formation occurs over a temperature range of 0
to 97°C, psychrophilic methane bacteria have not yet been isolated.7 The majority
of known methanogenic bacteria are mesophilic, with temperature optima around
450
35°C. A few extremely thermophilic methanogens have also been isolated.124 It
appears, however, that methanogenic activity is very low between 40 and 50°C.
Perhaps this temperature range is too high or too low for the mesophilic and
thermophilic methanogens, respectively. Two temperature ranges have been sug-
gested for the anaerobic digestion process: (1) mesophilic, 30 to 38°C; and (2)
thermophilic, 50 to 60°C. The advantages and disadvantages of these two tem-
perature ranges have been explored before. The reader is referred to a review by
Buhr and Andrews.125
In order to demonstrate the applicability of the Arrhenius model, the data on
the methanogenic activity of M. arboriphilus7'104 were used to fit the following
equation, which was developed based on the previously presented Hinshelwood
equation.
k = it, exp[a,(r - 30)] - k2 exp[a2(T - 30)] (27)
where k is the relative methanogenic activity. For T < 30°C, the second term of
Equation 27 is negligible and reduces to the following linear equation:
In it = In it, + at(T ~ 30) (28)
A plot of In it vs. (T — 30) resulted in a straight line with the following regression
equation: y = - 0 . 2 9 + 0.15 x (r2 = 0.982; n = 5). Therefore, it, = 0.75
anda, = O . ^ C " 1 . The values of it2anda2 were estimated by fitting the available
data and the resulting equation is
it = 0.75 exp[0.15(r - 30)] - 0.14 exp[0.30(r - 30)] (29)
Figure 6 shows the fit of the above equation. Buhr and Andrews125 used a similar
equation for the quantification of the effect of temperature on the net bacterial
growth rate (|x, d" 1 ) during thermophilic digestion:
H = 0.324 exp[0.06(r - 35)] - 0.02 exp[0.14(7 - 35)] (30)
Lawrence and McCarty89 found that the values of the yield coefficient (Y)
and the microorganism decay coefficient (b) during the conversion of volatile
fatty acids (i.e., acetic, propionic, and butyric) were nearly unaffected by tem-
perature. However, both the maximum specific substrate utilization rate (k) and
the half-velocity coefficient (K,) varied with temperature. The effect of temper-
ature on K, (mg/L) for acetic acid utilization was described by the following
equation (25°C =s T =s 35°C):
451
LU 10 20 30 40
OC
TEMPERATURE (°C)
FIGURE 6. Effect of temperature on methanogenic activity: (D) experimental data; line

according to Equation 29 (see text). (Data from Reference 104.)
O'Rourke17 developed similar equations for the quantification of the effect

of temperature on the biokinetic coefficients for primary sewage sludge digestion
(25°C ^ T ss 35°C):
(k)T = Ó. (32)
(KS)T = 2235[10 [OO46(35 -™] (33)
where k = maximum specific substrate utilization rate (d" 1 ); and Ks = half-

velocity coefficient (mg/L COD). Using the above correlation and assuming So
> Ks (the usual case for anaerobic digestion), the minimum solids retention time
as a function of temperature for primary sewage sludge digestion is given by the
following equation:
6?" = [0.267]lO'- 001« 35" 71] - 0.015]-' (34)
To arrive at the above equation, a yield coefficient equal to 0.04 g VSS/g COD
and a microorganism decay coefficient equal to 0.015 d ~ \ as proposed by
O'Rourke, were used.
452
Lin et al.126 investigated the temperature dependence of biokinetic parameters
for the methanogenesis of volatile fatty acids over a temperature range from 15
to 50°C. Chemostat-type reactors were used and the feed consisted of acetic,
propionic, and butyric acids at a ratio 2:1:1 (COD basis). The following corre-
lations were derived:126
(k)T = 7.4(1.077) (r - 25) (15°C ^ T < 35°C) (35)
(KS)T = 230(0.939) (r - 25) (15°C < T < 35°Q (36)
(Y)T = 0 . 0 2 ( 1 . ( W - 2 5 ) (25°C < T < 40°Q (37)
When the microorganism decay coefficient was neglected, the following

equation was developed:126
{ }
=
r 25)
0C 230[(0.939)< - ] + 5
For a given temperature, Equation 38 can be used to estimate either the effluent
substrate concentration (5) for a 6C value or to calculate the required 0C for a
desired substrate removal efficiency. Generally speaking, as the fermentation
temperature decreases, the effluent concentration increases. The most pronounced
effect of temperature is on the minimum solids retention time. Assuming So >
Ks, the following equation was developed for the dependence of 6™n on temper-
ature in the case of methanogenesis from volatile fatty acids:
G™" = [0.148(1.116)°"-25> - 0.015]- 1 (39)
The above equation is valid for a temperature range from 25 to 35°C. A mi-
croorganism decay coefficient equal to 0.015 d" 1 is used. The effect of temper-
ature on the minimum solids retention time for the case of anaerobic digestion
of primary sewage sludge and methanogenesis from volatile fatty acids based on
Equations 34 and 39, respectively, is shown in Figure 7. In the same figure, the
values of O^" at various temperatures as recommended by McCarty127 are also
shown. Based on the temperature dependence patterns shown in Figure 7, it
appears that the 6^" values suggested by McCarty are conservative. Nevertheless,
a safety factor of at least 2.5 is always applied for the estimation of the design
e c . 128
It is worth noting that, when the fermentation temperature was increased from
35 to 40°C, a sharp increase of the minimum solids retention time from 2.42 to
4.33 d was observed during methanogenesis of VFA.126 The minimum solids
retention time for the acidogenesis of glucose dropped from approximately 10 h
at 22.4°C to a minimum of approximately 2 h at 38°C and sharply increased to
approximately 7 h at 42°C.76 Another region with minimum solids retention time
453
12
McCarty (1964)
SRT (Days) 10
Z 4
Lin et al. (1987)
16 20 24 28 32 36
TEMPERATURE (°C)
FIGURE 7. Effect of temperature on the minimum solids retention time of the anaerobic
treatment process.
equal to 1.4 h at approximately 53°C was also observed. Based on the above
data, it is noteworthy that for soluble substrates the minimum solids retention
time for acidogenesis is in the order of hours whereas in the case of methanogenesis
it is in the order of days.
The optimum temperature for the acidogenesis of a complex substrate (beef
extract) was found to be 40°C.70 The Arrhenius equation adequately described
the temperature effect. The temperature coefficient (ß, 0 ) for the rate of acid
production between 30 and 40°C was equal to 1.29. With increasing temperature
from 25 to 40°C, an increase in acetic acid concentration was observed whereas
the concentration of other volatile fatty acids remained relatively constant.
Henze and Harremoes121 have compiled an extensive list of references related
to the temperature dependence of anaerobic processes. Based on seven sets of
data collected from the literature, these authors concluded that the optimum
temperature for mesophilic digestion is between 30 and 40°C. A plot of the natural
logarithm of the relative methanogenic activity (i.e., methane production rate at
a given temperature divided by the rate at 35°C) vs. temperature resulted in a
temperature coefficient equal to 0.10 o C~' in the temperature range 10 to 30°C.
Beyond 40°C, a precipitous decline of methanogenic activity was observed. This
temperature-methanogenic activity correlation agrees with the Hinshelwood model
(see Equation 26).
454
Westermann et al.129 reported more than a 40-fold increase of the half-sat-
uration constant (Km) with temperature increasing from 20 to 37°C in pure cultures
of Methanosarcina barker i (strain 227) grown either on H2/CO2 or acetate. The
maximum specific substrate utilization rate also increased with increasing tem-
perature. The temperature coefficient (Q10) was found to be substrate concentration
dependent: the lower the substrate concentration, the lower the Q10 value, thus
resulting in decreased temperature dependency. This response was termed "po-
sitive temperature modulation". This finding is in contrast with other reported
^-temperature correlations.17-126 Mass-transfer limitations might have been in
effect in the study carried out by Westermann et al.129 Since substrate utilization
rate increased with an increase in temperature, the effect of mass transfer was
probably more pronounced at the higher temperatures. In contrast, at low tem-
peratures, decreased concentration gradients exist since the substrate utilization
rate is low making mass transfer limitations less pronounced. The effect of sub-
strate concentration on the value of Ql0 can be shown by examining the specific
substrate utilization rate (U) at two different temperatures. According to Equation
6, it follows that:
010
-
where subscripts 1 and 2 refer to temperature 7, and T2 (for T2 = Tx + 10).

According to the temperature correlations developed by O'Rourke17 and Lin et
al.126 for primary sewage sludge digestion and methanogenesis from volatile fatty
acids, respectively, the dependence of Ql0 on substrate concentration is shown
in Figure 8. The "positive temperature modulation" of M. barkeri is also depicted
in Figure 8 according to data reported by Westermann et al.129
VI. KINETICS OF INHIBITION
Anaerobic processes, like all biological processes, are susceptible to inhibition

and toxicity brought about by various toxic substances. The degree of inhibition
depends on the nature and concentration of the toxic substance and whether or
not the biological system has been previously exposed to the toxic substance,
i.e., if acclimation has taken place. The toxic substances usually quoted for
anaerobic systems are ammonia, cations, heavy metals, sulfides, xenobiotics,
and volatile fatty acids.130'131 Excellent reviews and discussions of inhibition and
toxicity in anaerobic systems have previously been published.42'121-130"133 The
remainder of this section is devoted to the various kinetic expressions used to
model inhibitory effects. The inhibition and toxicity models used for anaerobic
systems can be divided into three categories: empirical, Monod-type with ad-
justable biokinetic constants, and the inhibition coefficient models.
455
Westerman et al. (1989)
O'Rourke (1968)
10 15
SUBSTRATE CONCENTRATION (g/L)

FIGURE 8. Effect of substrate concentration on Q10.
A. Empirical Inhibition Models
An empirical model for describing the recovery pattern from slug addition
of toxicants in methanogenic, acetate-fed systems has been developed by Parkin
and Speece:132
G, = Ae~k>' + Be**' (41)
where G, = methane production rate (ml/d); A, B = empirical constants (A +

B = control gas production); / = time after addition of toxicant (d);&i = toxicity
rate constant (d" 1 ); and k^ = recovery (or acclimation) rate constant (d" 1 )- This
model was successfully used to describe the recovery pattern for a wide variety
of toxicants such as cyanide, chloroform, formaldehyde, and copper. The recovery
pattern model adequately predicts the period of zero gas production. The threshold
and lethal doses can also be estimated.
B. Monod-Type Models with Adjustable Constants
The Monod equation for specific growth rate (and specific substrate utilization
rate) has been used for describing inhibition effects of toxicants in anaerobic
456
systems by allowing the four basic biokinetic constants (i.e., Y, Ks, k, and b) to
adjust depending on the type and concentration of the inhibitor.
Kugelman and Chin98 have reported on the effect of Na + and K + on the
kinetics of acetate utilization in chemostat-type reactors. Their results were pre-
sented as variable biokinetic coefficients as affected by each cation at various
concentrations. Based on the data of Kugelman and Chin,98 the effect of K + on
the kinetics of acetate utilization can be summarized as follows Y = 0.041 mg/
mg; b = 0.0356 d" 1 ; Ks = 161.4 mg/L. These coefficients were not affected
by the concentration of K + . However, the maximum specific substrate (i.e.,
acetate) utilization rate (k) decreased beyond a concentration of K + 5= 0.10 M.
The dependence of k on the concentration of K + can be expressed by the following
relationship, which is derived based on the data reported by Kugelman and Chin:98
k = 8.5 - 37[[K] - 0.1] 0.10 == [K] < 0.20 (42)
where [K] = molar concentration of K + (AÍ).

Increasing concentrations of sodium did not affect the values of Ks and k for
acetate utilization (i.e., Ks = 185 mg/L and k = 8.56 mg/mg-d), but both Y and
b were affected.98 The latter dependency can be expressed by the following
relationships:
Y = 0.054 - 0.08 [[Na] - 0.15] 0.15 < [Na] < 0.35 (43)
b = 0.065 + 0.305 [[Na] - 0.15] 0.15 < [Na] == 0.35 (44)
The antagonistic effect of sodium on the kinetics of acetate utilization in digesters

retarded by 0.2 M potassium was manifested as the reversal of the effect of
potassium on k: the value of k increased with increasing concentrations of sodium.
At the optimum sodium concentration of 0.03 M, the value of k was even higher
than in systems containing low potassium and no added sodium. Likewise, the
toxicity of 0.35 M sodium was reversed by potassium concentrations >0.03 M
by restoring Y and b to the values observed in digesters where sodium was <0.35
M.98
Another example of this type of toxicity modeling is the approach taken by
Parkin and Speece.I32 The values of both k and K, were adjusted to fit experimental
data of acetate-fed methanogenic systems inhibited by formaldehyde whereas Y
and b were constant. Their data are summarized in Table 16.
C. Inhibition Coefficient Models
An alternative to adjustable biokinetic "constants" in describing inhibition

and toxicity kinetics of biological systems is a series of models which are based
on the Monod equation with the incorporation of an inhibition correction factor.
In the case of reversible inhibition, three types of inhibition models have been
457
TABLE 16
Kinetic Constants for Acetate Utilization at 35°C a s Affected by
Formaldehyde Inhibition
Parameter value at
formaldehyde concentration (mg/L)
Parameter 0 100 250
Yield coefficient 0.04 0.04 0.04

(Y, mg/mg)
Half-velocity coefficient 700 850 400
(K,, mg/L)
Specific substrate utilization 5.2 3.7 1.7
rate
(k, mg/mg-d)
Microorganism decay rate 0.02 0.02 0.02

(b, d- 1 )
Washout retention time 6.9 10.8 25.5
1
(es* , d)
Note: Influent of substrate (i.e., acetate) concentration equal to 2700 mg/L.
Data of Parkin and Speece.132
proposed: competitive, uncompetitive, and noncompetitive.134 The specific sub-

strate utilization rate (U) can then be presented as follows:
Competitive:
~ (45)
K.[l+-)+S
Uncompetitive:
/ / (46)
Ks + S ( l + —
Noncompetitive:
kS
(47)
+
* ')('•*)
458
where / = concentration of inhibitor; and K¡ = inhibition coefficient (same
concentration units as I). Competitive inhibition affects the value of K„ i.e.,
(Ks)ap — Ks(l + I/K¡. Uncompetitive inhibition affects both k and Ks i.e., (k)ap
= Jt/(1 + I/K¡) and (Ks)ap = KJ{\ + I/K,). Noncompetitive inhibition affects
only k, i.e., (k)ap = k/(l + I/K,). The effect of the nondimensionalized substrate
concentration (S0/Ks) on the value of 8™n at various levels of the nondimension-
alized inhibitor concentration (I/K,) for the case of competitive and noncompetitive
inhibition is shown in Figure 9 for a CSTR reactor.
A special case of uncompetitive inhibition is the substrate inhibition where
the inhibitor and the substrate are the same substance. In this case, equation 46
for / = 5 leads to the Haldane equation:135
U= 1 r (48)
Another inhibition model that has been used for anaerobic systems is the gen-
eralized Haldane equation for substrate uncompetitive inhibition:136-137
where n = constant (determines the order of inhibition). When n = 1, Equation

49 reduces to the Haldane equation. The effect of SIKS on the relative specific
substrate utilization rate (i.e., Ulnh/k) is shown in Figure 10 for the special case
where K, = 3 Ks.
Dinopoulou et al.138 tested both the Haldane and the noncompetitive inhibition
models to describe the kinetics of acidogenesis in chemostat-type reactors using
a complex substrate (beef extract). Based on statistical analysis, the noncompe-
titive inhibition model was found to be superior to the Haldane model. The
estimated values of the biokinetic constants are as follows: maximum specific
growth rate ( f w ) = 32.6 d"1; half-velocity constant (iQ = 1.77 g/L (COD
basis); inhibition constant (/sT,-) = 0.633 g/L (total volatile fatty acids); and specific
decay rate (b) = 1.747 d " '. The specific product formation rate was also described
with the noncompetitive model. The maximum specific product formation rate
was equal to 125.7 g total VFA/g biomass/day.
Andrews and co-workers139-140 have used the Haldane equation to account for
substrate inhibition in anaerobic processes. In their case, the concentration of
free, undissociated VFA (mainly acetic and propionic) were considered to be the
substrate/inhibitor. Since the concentration of the undissociated VFA (mainly
459
30
CO
>»
CO
Û
20
CO
2
10
z
8 10
30
co
>»
(0
Û
20
J-
CO
2
10
I
8 10
FIGURE 9. Effect of substrate concentration on the minimum solids retention time in case
of inhibition. (A) competitive inhibition; (B) noncompetitive inhibition.
460
8 10
S/K,
FIGURE 10. Effect of the relative substrate concentration (S/Ks) on the relative specific
substrate utilization rate (U^/k) according to the generalized Haldane model (Equation 49;
K, = 3/CJ.
acetic and propionic) is related to the pH and pKa values, Equation 48 becomes
a function of these two parameters:
U = (50)
KJC.
1 +
where [H+] and [S] are the hydrogen ion concentration and total VFA concen-
tration, respectively (mol/L). The value of K¡ = 0.667 mA/ was used by Andrews
and Graef1 *° for the simulation of the anaerobic digestion process.
Hill and Barth141 extended the model developed by Andrews to account for
the inhibitory effect of ammonia in the case of anaerobic digestion of animal
waste:
U = (51)
+ +
VA Kla Ka
where VA = concentration of un-ionized acids (mg/L); Kia = inhibition coef-
ficient of acids (mg/L); NH3 = concentration of un-ionized (free) ammonia (mg/
L); and^TQ = inhibition coefficient of ammonia (mg/L). The following inhibition
constants were used by Hill and Barth:141 for methanogenic bacteria, Kn = 300
461
mg/L un-ionized VFA and Ka = 5 mg/L un-ionized NH3; for acidogenic bacteria,
K, = 1000 mg/L un-ionized VFA. An improvement of the model developed by
Hill and co-workers was presented where the inhibition of acidogenic and meth-
anogenic bacteria was accounted for separately and the microorganism decay rate
was expressed as a function of VFA.142
Lozano et al.143 used an expression resembling the noncompetitive model
(Equation 47) for describing the inhibitory effect of VFA in the acidogenesis step
of anaerobic digestion:
(52)
where/ = total concentration ofVFA(g/L); So = influent substrate concentration

(g/L); and a = a constant (inverse units of So).

Neufeld et al.137 applied the generalized form of the Haldane model (Equation
49) to describe the anaerobic degradation of phenol. Based on experimental data
where phenol was the only carbon source, the following values for the biokinetic
constants were obtained: K, = 700 mg/L; it = 0.08 g phenol/g VSS-d; K¡ =
966 mg/L; and n = 4.
Inhibitory effects of short-chain fatty acids and hydrogen gas in anaerobic
processes have largely been explained by the substrate and product inhibition
models. Fukuzaki et al.144 studied the methanogenic degradation of propionate
in a mesophilic (37°C) propionate-acclimatized anaerobic sludge reactor. Inhi-
bition of propionate utilization by propionate was described by the Haldane sub-
strate inhibition model with the following biokinetic constants: Ks = 15.9 |xM;
K¡ = 0.79 TOM; k = 2.15 mmol/g VSS-day. The substrate was expressed as the
concentration of undissociated propionic acid. The inhibition of propionate by
both hydrogen gas and acetate was described by a noncompetitive product in-
hibition model:144
(53)
1 + (—
where P = concentration of product (partial pressure of H2 and undissociated

acetic acid concentration, respectively); Kp = inhibition constant (same units as
P); and n = constant (inhibition order). The biokinetic constants derived from
data fitting by Fukuzaki et al.144 for the acetate-inhibited propionate utilization
are as follows: Kp = 48.6 \iM; k = 1.85 mmol propionate/g VSS-day; and n
= 0.96. For the hydrogen-inhibited propionate utilization, the following constants
were determined: Kp = 0.11 atm (71.5 \xM dissolved H2); k = 2.40 mmol
propionate/g VSS-day; and n = 1.51. It is noteworthy that the inhibition constants
of both hydrogen and acetate are in the same order of magnitude, thus suggesting
462
that the efficient removal of both products by methanogenic cultures is important
for maintaining high rates of propionate utilization.
Mosey145 used an expression similar to the noncompetitive model to describe
the effect of hydrogen gas on the degradation of propionic and butyric acids in
the anaerobic digestion process. A "regulator function" was derived by postu-
lating that, in reactions involving hydrogen transfer, the rate-limiting substrate
is the oxidized form of the electron carrier nicotinamide adenine dinucleotide
(NAD) according to the reaction:
NAD+ + H+ + 2e~ í± NADH (54)
Based on the redox potential of the NAD+/NADH pair ( - 3 2 0 mV at pH 7.0),

the following equation was developed by Mosey for the specific utilization rate
of propionic and butyric acids:145
kS
U =
(Ks + S)(l + 0.0015//)
where H = gas-phase hydrogen concentration (ppm, v/v); and 0.0015 is the

calculated redox constant. A detailed description of inhibition models can be
found elsewhere.69
VII. EFFECT OF MASS TRANSFER ON KINETICS
Aggregation of the methanogenic consortia in dense conglomerates retained

in the reactor leads to an uncoupling of the hydraulic and microbial (solids)
retention times. This key concept has resulted in the successful implementation
of anaerobic digestion for the treatment of a variety of wastes.'•146-147 The meth-
anogenic. aggregates are usually in the form of a biofilm (i.e., biomass attached
to support media) or are suspended and retained in the reactor by special solid-
liquid-gas separation structures. As the size of the aggregate increases, the surface
area-to-volume ratio decreases and the in-flux of substrates or the out-flux of
products may become the limiting step in the overall substrate removal kinetics.
In this section of the paper, the effect of mass transfer on the kinetics of substrate
uptake is discussed. In particular, the necessity for inclusion of mass transfer
expressions in the kinetic descriptions of substrate removal in methanogenic
microbial aggregates is addressed.
The various mass transfer steps involved during the degradation of organic
matter by bacterial aggregates are depicted in Figure 11. First, the substrate must
be transported from the bulk liquid across a stagnant liquid layer in the proximity
of the biofilm and then further transported to the surface of the aggregate. This
step is commonly termed "external mass transport". Then, the substrate must
diffuse throughout the aggregate matrix where it will be catabolized. Intermediate
463
blofllm bubbl»
support
gas channel
FIGURE 11. Generalized schematic of an anaerobic aggre-

gate showing the various mass transfer steps and the substrate
concentration profile.
products diffuse and react inside the aggregate, while final products must diffuse
out to the aggregate surface and finally to the bulk liquid. Mass transfer of
intermediate products can play an important role in the overall kinetics, especially
in the case of product inhibition as does hydrogen in acetogenic reactions and
acetic and propionic acids in acidogenic reactions.
A number of mathematical models for biofilms have been devel-
oped. 147a.I47b.147<: Suidan and Wang147d have developed simplified algebraic expres-
sions for biofilm kinetics, incorporating Monod-type substrate utilization rates
and diffusive mass transport. Suidan also developed a model for deep biofilm
reactors and presented nomograms which can be used to determine the perfor-
mance of biofilm reactors.147' Details of these and other biofilm models are not
presented in this article. The interested reader is referred to the literature for
further information.
A. External Mass Transfer Considerations
1. Soluble Substrates
During the transport of substrate from the bulk liquid to the surface of the
aggregate, different transport mechanisms need to be considered depending on
the size of the substrate. For soluble substrates (diameter <0.45 \im), the dom-
464
inant mechanism of transport is Brownian diffusion, whereas for paniculate sub-
strates, other mechanisms of transport, such as sedimentation and interception,
become important.148 Theoretically, if the flow field around an idealized aggregate
is known, the transport equation of the substrate from the bulk liquid to the
surface of the aggregate can be solved and the substrate flux can be obtained. In
real-life applications, due to the complex hydrodynamic regime involved, semi-
empirical correlations are usually used. A common problem encountered during
the application of these correlations is that they are usually obtained under con-
ditions different from those prevailing in anaerobic systems and they do not
account for the effects of the gaseous phase.
Recently, several correlations for mass transfer of soluble compounds in three-
phase (solid-liquid-gas), packed- and fluidized-bed reactors have been published.
Of special interest is the correlation proposed by Fukuma et al.149 where data
from previous studies were reviewed and incorporated into their results. The
proposed correlation is149
Sh = 2 + 0.51 ( * _ ) Sc113 (56)

\ v )
where Sh = Sherwood number = kPp/D; E = energy dissipation rate per unit
mass of liquid; 5c = Schmidt number ( = vID); k¡ = mass transfer coefficient;
Dp = diameter of the aggregate; D = diffusion coefficient; and v = kinematic
viscosity of water. This correlation holds true for packed- and fluidized-bed
reactors and it accounts for the effect of gas evolution on the mass transfer
coefficient. This correlation will be used for the estimation of the external mass
transfer coefficients in anaerobic reactors.
2. Paniculate Substrates
More often than not, the particulate fraction of wastewaters represents a

considerable fraction of the total oxygen demand. Particle deposition in biofilms
can interfere with the uptake of soluble substrates and can reduce the volumetric
activity of the biofilm. A significant amount of work on the kinetics of particle
transport has been done by investigators working in the area of water treatment.
A recent review by O'Melia150 summarizes the problem. In contrast to the soluble
substrates where convection and diffusion are the main mechanisms of substrate
transport, it is necessary in the case of particulate substrates to consider two
additional mechanisms: gravity sedimentation and interception. 148>150-151 The math-
ematical description of these mechanisms is as follows:
£ + WS = DW +(l- *-)(^r) - (57)
465
where v = fluid velocity vector; p, pp = water and particle density, respectively;
m = mass of the particle; g = gravitational acceleration; and |x = water viscosity.
Equation 57 is derived from a mass balance on 5 in an elemental volume of
suspension. The term v V 5 represents the advection transport, the term D WS
represents the transport by diffusion, and the last term on the right-hand side
represents transport by sedimentation.152 Interception transport is accounted for
mathematically as a boundary condition. Equation 57 cannot be solved analyti-
cally, therefore numerical methods and/or simplifying assumptions must be used.151
It must be noted that Equation 57 does not consider the hydrodynamic retardation
effect, which accounts for the combined effects of electrostatics, van der Waals
forces, and hydrodynamic interactions that occur when two particles approach
each other within a distance of a few radii.150 O'Melia has presented applications
of the above equation to filtration, deposition of particles in pipelines, floccu-
lation, sedimentation, and fate of particles in lakes.148 More recently, Bouwer152
applied the concepts of particle transport to biofilm-type reactors. Due to the

complexity of the application, he used semiempirical correlations developed by
other investigators in the area of water filtration to estimate the rates of particle
transport in different biofilm reactors. As pointed out by Bouwer, the results are
based on well-defined velocity gradients and boundary layers near the biofilm.
These conditions may not necessarily hold true for anaerobic aggregates, where
continuous gas evolution and surface roughness can create hydrodynamic con-
ditions which are far from well defined.
An additional source of concern is the interaction between the physical trans-
port of the particle to the surface of the biofilm and the chemical attachment of
the particle onto the biofilm. In water treatment applications, particles are chem-
ically destabilized prior to filtration. However, this is not the case in wastewater
applications where the ratio of collisions that result in successful attachment to
the total number of collisions, the so-called collision efficiency factor (a), is
expected to be in the range of 0.001 to 0.1. l 4 8 Unfortunately, when the chemistry
is unfavorable, the predictive capacity of this approach decreases due to specific
adsorption phenomena and the lack of adequate descriptions for the three-di-
mensional structure of the solid-water interface. 15°
Recently, Sprouse and Rittmann153 have presented experimental data on the
removal of colloidal particles from a milk solution in an anaerobic fluidized-bed
reactor. The majority of the particles were in the 0.45 to 2.0-|xm size range. A
75% removal efficiency based on total suspended solids was obtained; however,
since some of the effluent particles were biomass, this removal efficiency does
not accurately represent the performance of the reactor. After subtraction of the
biomass fraction, the particle removal efficiency was calculated to be 90%. Based
on a theoretical model, the aforementioned authors concluded that recirculation
contributes significantly to particle removal in fluidized-bed reactors. Bed ex-
pansion did not affect the particle removal for a given organic load, and higher
concentrations of influent suspended particles increased the overall particle re-
moval efficiency. The estimated a was 0.04 which agrees with values expected
in wastewater applications.148
466
Once the particles are adsorbed onto the biofilm surface, they must undergo
hydrolysis. A comparison of the rates of hydrolysis and the rates of particle
attachment for a given particle size would give some insight as to which process
is rate-limiting. However, with the available data, it is not possible to make this
kind of comparison. Based on the preceding discussion, it seems that our un-
derstanding of the kinetics of paniculate substrate removal in biofilms is still
incomplete for engineering applications, and more research is necessary.
B. Internal Mass Transfer Considerations
The transport of substrates inside biological aggregates has been traditionally

modeled using Brownian diffusion as the only transport mechanism. Recently,
Logan and Hunt154 have proposed that advective transport plays a significant role
in the transport of soluble substrates in highly porous aggregates such as those

usually found in aerobic environments. In anaerobic digestion applications, the
aggregates have densities several times higher than those found in aerobic ap-
plications. Consequently, the mechanism for internal mass transfer (i.e., inside
the biological matrix of the aggregate) will be considered to be diffusion only.
A possible exception is the light aggregates often observed in acidogenic reactors.
In the case of diffusional mass transfer, Fick's law applies:155
J--D% (58,
where J = flux; D — diffusion coefficient; S = solute concentration; and x =

system's coordinate. The diffusion coefficient is usually considered constant or
independent of the solute concentration. D is not an easily measurable parameter
and often its value is obtained by multiplying the diffusion coefficient of the
solute in water (Dw) by an empirical constant usually smaller than unity. Another
approach is to use the diffusion coefficient as a fitting parameter for an observed
set of data. The ratio of diffusion coefficients (i.e., D/Dw) in aerobic biological
matrices is in the range of 0.1 to I.156-157 Limited information exists on the
diffusion coefficient of solutes in anaerobic biological matrices. The available
information is summarized in Table 17. Both sets of data reported in Table 17
were obtained using artificially established aggregates, thus the influence of the
actual structure of the methanogenic aggregate was neglected. The importance
of the influence of this structure on the effective diffusion coefficient is not known,
but one can expect that permanent gas evolution through observed gas channels
would impose a significant barrier to solute transport inside the aggregate. In any
case, the available information suggests that the diffusion coefficients for solutes
inside the methanogenic aggregates are between 10 and 30% of those found in
clean water. These values are in contrast with the D/Dw ratio of 0.80 as determined
by Williamson and McCarty160 for artificially established nitrifying biofilms.
The presence of small gas bubbles on the surface of methanogenic aggregates
467
TABLE 17
Diffusion Coefficients in Methanogenic
Aggregates at 35°C
D
Compound (cm 2 /s) D/Dw Ref.
Ethanol 1.20 x 10~6 0.12 158

Acetate 1.71 x 10~6 0.12 158
Hydrogen 1.39 x 10~5 0.27 158
Lithium — 0.22-0.33 159
Note: Temperature corrections were made according to

the Einstein-Stokes formula.
is ubiquitous. Several researchers have concluded that gas effervescence in an-

aerobic biofilms is responsible for biofilm sloughing and granule bursting, es-
pecially during overload conditions.161~164 Henze and Harremoes'21 have pointed
out the importance of this last step in anaerobic biofilms and suggested that the
outward diffusion of gases in the form of bubbles would create enough disturbance
to alter the whole diffusional pattern in the film. Surprisingly, there has not been
any additional work on the effect of the transport of gaseous products on the
performance of anaerobic biofilms. Electron microscopy of anaerobic aggregates
reveals the presence of volcano-type structures at the surface of the aggregate,
and it is believed that these structures function as gas evacuation outlets164"167
The simple presence of bubbles in the biofilm implies the existence of mass
transfer limitations. If the outward transport of gases is slower than their biological
production, gas will accumulate and lead to supersaturation in the biofilm matrix.
In this case, a bubble would form and the aggregate would burst or the biofilm
would slough off. The presence of gas channels inside the biological matrix has
been documented by electron microscopy.164'165 It is worth noting that the diffusion
coefficient of gas in gas (e.g., carbon dioxide in methane or vice versa) is roughly
105 times higher than the diffusion coefficient of gas in liquid.155 Following this
reasoning, one realizes that the gas channels inside the biological matrix may act
as efficient drainage structures for gaseous products by bringing the gaseous
products from the deep part of the aggregate to the surface. Bubble formation at
the surface of the aggregate would therefore reduce the biofilm area available for
external transport (i.e., from the biofilm surface to the bulk liquid). More research
is necessary to arrive at a better understanding of the impact of the transport of
gaseous products on the kinetics of substrate utilization in methanogenic aggregates.
468
C. Comparison of Mass Transfer and Substrate Utilization Rates
1. Observed Mass Transfer Rates in Methanogenic Systems
There have been few studies in which the question of mass transfer effects
on the observed kinetics of anaerobic aggregates has been directly addressed.
Finney and Evans168 argued that the rate-limiting step in the overall process is
the outward diffusion of gases. Initial evidence for their conclusion was the low
temperature dependence of the methanogenic step, typical of transport-limited
processes. A sixfold increase in the turnover of acetate, as compared to the data
of Lawrence and McCarty,89 was achieved under subatmospheric pressure and
vigorous agitation. Implicit in their conclusion is the assumption that methano-
genesis is product-inhibited. Dolfing169 studied the effects of the size of granules
from Upflow Anaerobic Sludge Blanket (UASB) reactors on the observed kinetics
of formate, hydrogen, propionate, and acetate at 30°C. Significant mass transfer

effects were observed for formate, hydrogen, and acetate. Smaller but insignif-
icant effects were observed for propionate. Wang et al.170 concluded that no
significant external or internal mass transfer effects existed in the kinetics of
acetate consumption in a fluidized-bed methanogenic reactor at 35°C subjected
to low loading. Consequently, this reaction could be modeled with Monod kinetics.
Hamoda and Kennedy162 observed half-order reaction kinetics for acetate
removal in a Down-flow Stationary Fixed Film (DSFF) reactor at 35°C. Half-
order reaction kinetics are typical of mass transfer-affected biological kinetics.157
Kennedy and Droste171 concluded that there was no diffusional limitation in the
kinetics of a DSFF reactor treating a carbohydrate waste at 35°C with a biofilm
thickness of 0.9 to 2.6 mm. This conclusion was reached after comparison of
specific biofilm activities with respect to biofilm thickness. Canovas-Diaz and
Howell172 observed higher methane production rates in a comparison of DSFF
reactors and an anaerobic trickling filter (ATF). They attributed their results to
the better mass transfer characteristics for the release of gaseous products in the
ATF reactor.
Noyola et al.173 observed that the behavior of a rotating stationary fixed-film
reactor treating settled domestic sewage was highly dependent on the rotational
velocity, concluding that, at low temperatures (i.e., 16°C) and with domestic
sewage, external mass transfer is the rate-limiting step. This effect was not
observed at higher temperatures (i.e., 29°C). Denac and Dunn,174 comparing the
performance of packed- and fluidized-bed reactors treating molasses and whey,
concluded that the superior performance of the fluidized-bed reactor at high COD
influent concentration (i.e., higher than 3 g COD/L) is probably due to diffusion
limitations and lower biomass activities in the packed-bed reactor. The existence
of mass transfer limitations is not clearly demonstrated. Results of specific sludge
activity dependence on temperature and recirculation flow using Expanded Gran-
ular Sludge Bed (EGSB) reactors were presented by de Man et al.175 A low
temperature dependence of the specific sludge was observed, and there was no
effect by the recirculation ratio. Results of the effect of recirculation flow rate
469
on the calculated specific sludge activity of a UASB reactor treating molasses at
low temperature (i.e., 8°C) were also presented by the same authors. Significant
effects were observed with the increase of the recirculation ratio up to 1.3, whereas
a further increase to 3.0 did not exhibit any additional improvement. These results
were attributed to better sludge-water contact. Beeftink and Staugaard176 dem-
onstrated the existence of mass transfer effects in anaerobic microbial aggregates
growing on carbohydrates. This result was obtained by observing the spatial
arrangement of cells in the aggregates. Active cells were located in the outside
portion of the aggregates, while the center of the aggregates was composed of
lysed cells and exopolymers.
The dependence of the observed substrate utilization rates on temperature can
be related to the mass transfer limitations encountered in catalyzed reactions. The
basic idea is that diffusional and convective transport of solutes show a mild
temperature dependence compared to the biological reactions. For more details
concerning chemical catalysis, the interested reader is referred to a textbook on

this subject.177 The mathematical description of temperature dependence of meth-
anogenesis was presented in an earlier section of this paper. Figure 12 presents
the typical observed temperature dependence of methanogenic activity for dif-
ferent high-rate anaerobic reactors.178-181 In general, anaerobic reactors show a
lower temperature dependence than that observed in methanogenic cultures (see
130
117
104
91
ü
78
O 65
LU 52
39
26
<
13
LU 0
15 25 35 45
TEMPERATURE (°C)
FIGURE 12. Effect of short-term temperature variations on the relative methanogenic
activity of different reactors. (Data for UASB from Reference 179; Clarigestor from Reference
180; DSFF from Reference 178; and fluidized-bed from Reference 181.)
470
Figure 6). Fluidized-bed and DSFF reactors show the lowest temperature depen-
dence indicating the presence of mass transfer limitations. For UASB reactors,
the methanogenic activity/temperature curve exhibits a double dependence below
and above 25°C. This phenomenon is typical of internal mass transfer
limitations.177-182
From this brief overview of the observed phenomena related to the kinetics
of mass transfer in methanogenic reactors, it can be concluded that with few
exceptions the evidence for the significance of mass transfer effects in the different
reactor configurations is circumstantial and, in some cases, contradictory.
2. Estimation of the Extent of Mass Transfer Effects in Anaerobic

Reactors
The mathematical expressions necessary to make a theoretical estimate of the

extent of mass transfer effects on the observed kinetics of anaerobic reactors is
presented below. For the purpose of this analysis, the phenomena of aggregation,
aggregate growth, decay, and sloughing are not considered herein. It will be
assumed that the following characteristics of the aggregate are known: size,
kinetics of substrate utilization, shape (sphere), and composition (homogeneous).
Transport inside the aggregate is described by Fick's law and the diffusion coef-
ficient is independent of the solute concentration. Partition of substrate between
the liquid and the biological phase is not considered and no product inhibition
occurs. External mass transfer is described using the film theory, thus the gradient
of substrate concentration outside the particle is considered linear.
The steady-state transport equation for diffusion and reaction inside a con-
glomerate of spherical shape is derived from a mass balance of the solute on an
infinitesimal shell in the matrix, and is expressed as follows:155
Ad2
A rdr)d) Km
The two boundary conditions are as follows: at r = Ro, dS/dr = 0; and at r =
Rp, Js = Deff dSldr = k¡ (S, - Ss); where S = substrate concentration; D^ =
effective diffusion coefficient in the biological matrix; r = radial distance from
the center of the particle; RB, Rp = radius of the support particle and the coated
particle, respectively; V^ = maximum substrate uptake rate per unit volume of
biofilm; and Km = half-saturation constant. The subscripts / and s refer to the
bulk liquid and the biofilm surface, respectively. Other terms are as previously
defined. A schematic of the concentration gradients in the aggregate is presented
in Figure 11.
Equation 59 can be conveniently expressed in terms of nondimensional groups
as follows:
471
4>2ß
T^ = 0 (60)
The two boundary conditions become: at x = 1, xo, dfi/dx = 1; and at x = 1,

dßldx = 0.5 Sh (ß, - ß j ; where x = rlRp; ß = dimensionless substrate
concentration = S/Km; $ = Thiele modulus = (RlVmJKm D) 05 ; and Sh =
2 kfiJD. Other symbols are as previously defined. Equation 60 has been integrated
by numerous authors using numerical methods and approximations. The interested
reader is referred to literature for specific information.182"186 A common means
of expressing the results of the integration of Equation 60 is accomplished by
employing the overall effectiveness factor On,), which is defined as the ratio of
the observed (diffusion-limited) substrate uptake rate to the rate that would have
occurred if there were not significant diffusion limitations.
The effect of external mass transfer limitations on the observed substrate

uptake rates is usually evaluated using a modified Damkoehler number: Da =
V'^Jk^Cm, where V^ = maximum substrate uptake rate per unit of aggregate
surface area. For a spherical particle of diameter Dp, Da = V^J)/^ k,Km).
External and internal mass transfer limitations are combined in an overall effec-
tiveness factor, which will be evaluated for different situations.
Estimates of potential mass transfer rates in different reactors was developed
based on the above-presented equations. Reactor characteristics used for these
calculations are presented in Table 18. External mass transfer coefficients were
estimated using these values and the correlations recently developed for three-
phase systems (gas-liquid-solid) by Fukuma et al.,149 which were presented in an
TABLE 18
Range of Parameters Used in the Determination of External Mass
Transfer Effects for Different Anaerobic Reactors
Type of Reactor
Parameter* UASB Hybrid Filter Fluidized DSFF
v,(m/h) 0.5-2.0 0.5-3.2 0.06-3.6 8-20 5-7

vB(m/h) 0.12-2.7 0.05-4.8 0.12-2.7 0.12-15 0.1-5.5
e (dimensionless) 0.4-0.5 0.4-0.5 0.4-0.5 0.4-0.8 0.92
Dp (mm) 0.5-5.0 0.5-5.0 0.1-1.2 0.1-2.6
n (dimensionless) 3.25-4.35
D (m2/s) 1.0 x 10- 9
Xv (g VS/m!) 0.07-0.1 0.07-0.1 0.07-0.1 0.105-0.4 0.06
L (a COD/aVS-d) 0.34-1.9 0.85-1.05 0.3-2.4 0.9-1.4
v, = liquid velocity (includes recirculation flow); vg = gas velocity; e = porosity; Dp

= particle diameter; n = coefficient of the Richardson-Zaki equation {v = v,e" where:
vand v, = actual and terminal velocity, respectively); D = diffusion coefficient (depends
on the compound); Xv = concentration of volatile solids per volume of biofilm; Ls =
specific loading rate (temperature varies from 25 to 35°C).
472
earlier section. These correlations have the advantage of incorporating the effect
of gas generation on the mass transfer coefficient, and they were developed over
ranges of parameters that include those observed in anaerobic reactors. One
important result pointed out by Fukuma et al.149 and observed during the course
of this exercise is the importance of the gas phase on the mass transfer coefficient.
For small liquid flow velocities, gas flows are responsible for the majority of the
transport action. This result is very important for the development of scaling
criteria from laboratory and pilot studies. Superficial gas flow rates increase
linearly with the height of the reactor, thus mass transport characteristics improve
with increased reactor size.
The range of mass transport parameters for commonly used anaerobic reactors
were estimated and the results are summarized in Table 19. For the purpose of
correlation, the reactors were grouped into two categories: packed-bed and flui-
dized-bed. Packed-bed reactors include anaerobic filters, UASB, and hybrid re-
actors. The fluidized-bed group included EGSB, expanded, and fluidized-bed

reactors. The calculated Reynolds numbers for the anaerobic reactors are typical
of the Stoke's regime (Re =s 5.8) with few exceptions that follow Allen's re-
gime.187 In general, mass transfer coefficients in fluidized-bed reactors are higher
than those in packed-bed reactors. This is due to a combination of several factors
such as the tall, thin geometry of the fluidized-bed reactors (i.e., higher liquid
flow rates, higher gas flow rates) as well as the smaller particle sizes in these
reactors.
Calculated external mass transfer coefficients were combined with substrate
uptake kinetic parameters reported in the literature to calculate the Damkoehler
number for external mass transport and the generalized Thiele modulus for Monod
kinetics. These parameters were then used to calculate the overall effectiveness
factor for Monod-type kinetics for a given value of the parameter SIKm. This was
done for different substrates considering a best and a worst case scenario. The
TABLE 19
Range of External Mass Transfer Parameters for
Anaerobic Reactors
Parameter*
k,
Reactor type Re Sh (m/s x 10-5)
Packed bed" 0.4-21 13-120 0.6-1.6

Fluidized bed" 0.4-3.0 12-180 1.0-13.0
• Re = Reynolds number; Sh = Sherwood number; k, = mass

transfer coefficient.
" Anaerobic filters, UASB, and hybrid reactors are considered as
packed-bed reactors.
c
EGSB reactors are considered as fluidized-bed reactors.
473
results are summarized in Tables 20 to 23. During the following discussion, an
overall effectiveness factor <0.7 indicates significant mass transfer limitations.
In addition, it should be noted that external mass transfer does not become
significant until Da is > 1 for low substrate concentrations (i.e., concentrations
smaller than the half-saturation constant) and > 3 for substrate concentrations
higher than the half-saturation constant.188
Data presented in Tables 20 to 23 indicate that, in general, external mass
transfer resistances become important when internal resistances are already sig-
nificant. External mass transport limitations are not significant for acetate or
propionate uptake in aggregates up to 2 mm in diameter for packed- and fluidized-
bed reactors over a wide range of bulk substrate concentrations. For higher
aggregate diameter and for low bulk substrate concentrations, external mass trans-
fer becomes important for acetate in packed-bed reactors. The situation is different
for glucose and hydrogen. In packed-bed reactors, for all sizes of aggregates,
external mass transfer becomes highly important. For fluidized-bed reactors, using
glucose as the substrate, significant effects are expected at low substrate con-
centrations. Although hydrogen is not likely to be an intermediate substrate ex-
changed through the bulk liquid phase, the aforementioned mass transfer limi-
tations should be taken into account when exogenously supplied hydrogen gas is
used for the measurement of the hydrogen half-saturation constant. In this case,
the measured half-saturation constant will be much higher than the true intrinsic
value. Internal mass transfer resistances in packed-bed reactors are expected to
be significant for acetate at all substrate concentrations of interest for aggregate
TABLE 20
Theoretical Evaluation of Mass Transfer Resistance for the Utilization of
Acetate in Anaerobic Reactors
Case*
Parameter" 1 II III IV V VI VII VIII IX
Independent
Dp (mm) 0.5 0.8 1.0 1.0 5.0 0.5 0.5 1.0 5.0
ß (dimensionless) 1.0 0.2 1.0 0.2 1.0 1.0 0.2 0.2 0.2
Dependent
Sh (dimensionless) 30 41 48 48 164 17 17 26 89
k, (m/s x 10- 5 ) 1.90 1.86 1.74 1.74 1.20 1.2 1.2 0.9 0.6
Da (dimensionless) 0.1 0.2 0.3 0.3 2.0 0.2 0.2 0.5 3.6
<f> (dimensionless) 2.2 3.5 4.4 4.4 22.0 2.2 2.2 4.4 22.0
ti (dimensionless) 0.96 0.69 0.73 0.59 0.19 1.0 1.0 0.6 0.1
Cases I through V and IV through IX were calculated for high and low fluid and gas flow
rates, respectively.
Dp = particle diameter; ß = dimensionless substrate concentration; Sh = Sherwood
number; k, = mass transfer coefficient; Da = Damkoehler number; 4> = Thiele modulus;
•n = effectiveness factor.
474
TABLE 21
Theoretical Evaluation of Mass Transfer Resistance for the Utilization of
Propionate in Anaerobic Reactors
Case*
Parameter" I II III IV V VI VII VIII IX X
Independent
Dp (mm) 0.5 1.0 2.0 2.0 5.0 0.5 1.0 1.2 2.0 5.0
ß (dimensionless) 1.0 0.2 1.0 0.2 1.0 0.2 0.2 0.2 0.2 0.2
Dependent
Sh (dimensionless) 31 50 84 84 171 17 28 31 46 93
k, (m/s x 10~5 2.0 1.6 1.3 1.3 1.1 1.1 0.9 0.8 0.7 0.6
Da (dimensionless) 0.04 0.1 0.3 0.3 0.8 0.08 0.2 0.2 0.5 1.4
<J) (dimensionless) 1.4 2.8 5.6 5.6 14 1.4 2.8 3.4 5.6 14.1
TI (dimensionless) 1.0 0.8 0.6 0.5 0.3 1.0 0.8 0.7 0.5 0.2
• Cases I through V and IV through X were calculated for high and low fluid and gas flow rates,
respectively.
b
Dp = particle diameter; ß = dimensionless substrate concentration; Sh = Sherwood number; k, =
mass transfer coefficient; Da = Damkoehler number; <)> = Thiele modulus; t\ = effectiveness factor.
TABLE 22
Theoretical Evaluation of Mass
Transfer Resistance for the
Utilization of Hydrogen in Anaerobic
Reactors
Case
Parameter 1 II
Independent
Dp (mm) 0.5 5.0
ß (dimensionless) 0.2 0.2
Dependent
Sh (dimensionless) 30 89
k,(m/s x 10-5) 8.1 2.4
Da (dimensionless) 30.8 1035
$ (dimensionless) 37.3 373
T| (dimensionless) 0.09 0.0006
Dp = particle diameter; ß = dimensionless

substrate concentration; Sh = Sherwood
number; k, = mass transfer coefficient; Da
= Damkoehler number; <(> = Thiele mod-
ulus; -i) = effectiveness factor.
475
TABLE 23
Theoretical Evaluation of Mass Transfer
Resistance for the Utilization of Glucose in
Anaerobic Reactors
Packed-bed
reactors Fluidized-bed
Parameter Casel Case II reactors
Independent
Dp (mm) 0.5 5.0 1.12
ß (dimensionless) 1.0 1.0 1.0
Dependent
Sh (dimensionless) 30.1 164 76
k, (m/s x 10-5)
0.9 0.5 1.0

Da (dimensionless) 25 465 50
<>
| (dimensionless) 33 337 76
T) (dimensionless) 0.1 0.01 0.06
Dp = particle diameter; ß = dimensionless substrate con-

centration; Sh = Sherwood number; k, = mass transfer
coefficient; Da = Damkoehler number; <> j = Thiele mod-
ulus; T) = effectiveness factor.
sizes >0.8 mm in diameter. For propionate, aggregate sizes >1.2 mm in diameter

show significant mass transfer resistance.
The results from the theoretical evaluation of mass transfer compare well
with some of the experimental observations. For example, for UASB reactors,
recirculation does not seem to improve the observed specific substrate utilization
rate as reported by de Man et al.175 This result follows from the comparison of
external mass transfer rates and substrate utilization rates in Tables 20 and 21
(see Da number). Recirculation may have other beneficial effects such as better
water-sludge contact. It can be argued that fluidized-bed reactors exhibit better
performance than packed-bed reactors as a result of better mass transfer char-
acteristics. From the viewpoint of external substrate transfer, our results suggest
that this might be the case for carbohydrates and hydrogen, but it is not necessarily
true for acetate and propionate since the transport velocities for these substrates
are high enough to avoid external mass transfer limitations. Other phenomena
not considered here, such as product inhibition, might also play an important role
when coupled to mass transfer effects.
Glucose kinetics are highly affected by mass transfer limitations, both internal
and external, as evidenced by cell counts of acidogenic aggregates which showed
the viable cells to be distributed in a thin ring around the perimeter of the
conglomerate.176 The results presented by Droste and Kennedy183 for carbohydrate
wastewater treatment by a DSFF reactor seem to be in contradiction to the previous
results of Hamoda and Kennedy162 for acetic acid wastewaters as well as the
476
general trends revealed by our calculations. During the treatment of acetic acid
wastewaters in DSFF reactors with a biofilm thickness of 2 mm or less, a half-
order kinetic relationship was observed, which is typical of mass transfer limited
systems.157 The specific substrate utilization rates observed in this study were
similar to those observed in the carbohydrate wastewater study. Nonetheless, in
the latter study, one of the conclusions reached was that the biofilm was not
affected by mass transfer limitations up to a thickness of 2.6 mm. Since degra-
dation of carbohydrates often exhibits faster kinetics than acetate, these two
conclusions are contradictory. A possible explanation can be the unique char-
acteristics of the biofilm reported in the carbohydrate wastewater study. The
reported nominal biofilm thickness was measured after the biofilms were removed
from the liquid which led to a collapse of the biofilm. In the liquid, the biofilm
had tentacle-like appendages and were buoyant, which probably enhanced mass
transfer to a significant extent.188a Another possibility may be that the carbohydrate
fermentation actually occurred in the bulk liquid and was mediated by microor-
ganisms growing in suspension, as reported by Murray189 for an analogous lab-
oratory system treating bean-blanching waste. Similar observations showing the
acidogens growing in suspension and the methanogens growing attached on the
support medium have also been reported for fluidized-bed reactors treating an
artificial glucose wastewater.190
Hydrogen uptake rates are also strongly affected by mass transfer limitations.
This result implies that steep concentration profiles will develop inside the ag-
gregate when hydrogen is supplied exogenously. Although it is unlikely that
hydrogen will be exchanged through the bulk liquid (it is rather generated and
consumed inside the aggregate under normal conditions), the existence of steep
profiles helps explain the absence of inhibition in the degradation of propionate
which has been observed by several researchers in apparent contradiction to
thermodynamic calculations.4-174-191 According to thermodynamic calculations,
the free energy change for the degradation of propionate turns positive at ap-
proximately 10" 4 aim.1 Nonetheless, the exogenous measured hydrogen partial
pressures necessary to stop propionate degradation by hydrogen were much higher
than the aforementioned thermodynamic limit.4-174-191 McCarty and Smith146 re-
ported inhibition of propionate degradation by hydrogen in accordance with ther-
modynamic calculations when hydrogen was supplied through ethanol. In this
case, hydrogen was provided by a precursor (ethanol) and was generated inside
the aggregates.
3. Effect of Mass Transfer on the Observed Substrate Uptake Rates
The problem of mass transfer effects on the observed kinetics of substrate

uptake has been considered in detail by several authors and the interested reader
is referred to the literature.185-188-192 A brief discussion of the effects of mass
transfer on the observed substrate uptake rates will be presented here.
477
Based on our definition of the effectiveness factor (T|), the observed, mass
transfer-limited substrate uptake rate (Vo) can be expressed as:
(61)
where V,- = substrate uptake rate in the absence of mass transfer limitations; and
5 ; = substrate concentration in the bulk, liquid phase. All other symbols are as
previously defined. Equation 61 is linearized and usually used to obtain a double
reciprocal plot according to the following equation:
m
Since Tj is a function of S¡, a straight line should not be obtained, particularly if

a wide range of substrate concentrations is tested.185 Nonetheless, if a straight
line is fitted, an apparent half saturation constant is obtained that relates to the
intrinsic one as: K'm = KJt\. Thus, the value of the apparent half-saturation
constant would increase as the mass transfer limitations become more severe.
This effect has been experimentally observed by Dolfing169 in his analysis of
the kinetics of acetate, propionate, formate, and hydrogen in granular sludge from
several UASB reactors. Following this line of reasoning, it is expected that studies
conducted with exogenously supplied hydrogen have overestimated the intrinsic
half-saturation constant for hydrogen uptake. Giraldo-Gomez et al.,111 using an
experimental protocol that avoided mass transfer limitations (i.e., hydrogen was
supplied through propionate degradation), evaluated the half-saturation constant
for hydrogen uptake as equal to 4.3 mg/L in the gaseous phase (equal to 6 ng/L
as dissolved hydrogen). This value is about three orders of magnitude smaller
than previously reported hydrogen half-saturation values (see Table 11).
It is important to comment at this point about the results of Contois,33 who
observed that the growth rate of a culture was a function of the population density
(Equation D, Table 2). He arrived at the dependence of the half-saturation constant
on population density based on analysis of double reciprocal plots. One might
now suspect the existence of mass transfer limitations during these types of
experiments. From a practical perspective, Contois kinetics.provide a simple
alternative means to model the effect of mass transfer in anaerobic aggregates.
Orozco,193 working with activated sludge reactors, observed a similar dependence
of the half-saturation constant on the population density and proposed a similar
relationship. Contois kinetics have been proposed by Chen and Hashimoto35-194
and Orozco and Noguera195 to describe substrate uptake kinetics in methanogenic
reactors. As mentioned earlier, the Contois kinetic expression predicts a depen-
dence of the effluent substrate concentration on the influent concentration, a result
typical of mass transfer limited reactors.
Another approximation of the kinetics of transfer-limited biological reactions
478
is that of the half-order kinetics.157 It can be theoretically shown that zero order
reactions turn into half-order reactions when mass transfer limitations become
significant. The interested reader is referred to a review of biofilm kinetics by
Harremoes for a detailed description.157 When the Thiele modulus for the biofilm
(<j)) is > 4 and the parameter ß (i.e., S/Ks) is > 5 , the half-order approximation
for biofilm kinetics is considered satisfactory. It can be seen from Tables 20
through 23 that this approximation thus holds true for high acids concentrations
and aggregates > 1 mm, in the case of acetate, and > 2 mm for propionate. This
type of dependence has been observed for acetate consumption in DSFF reactors.162
4. Mass Transfer of Intermediate Metabolites
Due to the multistep nature of the anaerobic degradation of organic matter, '
metabolites produced by one microbial group during an early stage of the deg-
radation have to be transported to the following microbial group in the chain.
This latter step implies the development of concentration gradients between the
different bacterial populations. Because of the complex energetic relationships
involved, concentration gradients may have important effects on the behavior of
the microbial ecosystem.
One of the most studied intermediate metabolites is H2 because of its regu-
latory effects on the anaerobic degradation of organic matter, as previously dis-
cussed. Gujer and Zehnder,8 based on the mean residence time of hydrogen in
an anaerobic digester, calculated that syntrophic mircoorganisms must be, on the
average, closer than a distance of 76 |xm. Conrad et al.196 proposed that inter-
species hydrogen transfer occurs between juxtapositioned microbial associations
within the floes, and that just a small percentage of the total hydrogen transfer
is exchanged with the external (outside the floe) hydrogen pool. To account for
the observed volumetric COD removal rates, McCarty and Smith146 calculated,
based on typical volumetric loading rates for high rate anaerobic reactors and a
one-dimensional diffusion equation, that distances between hydrogen-producing
and -consuming microorganisms must be 5 to 10 fun. Prensier et al.197 have used
monoclonal antibodies to examine syntrophic microbial associations in granules.
Their observations showed very close associations (<50 nm) in the case of
Syntrophobacter. Thus, it is well established that very close associations favor
interspecies metabolite transfer, the apparent reason being the minimization of
interspecies diffusional gradients for intermediate metabolites (e.g., H2, formate,
acetate).111-146-197
Recently, Thiele et al.198-199 have proposed that formate, instead of hydrogen,
is the electron carrier between syntrophic microorganisms degrading ethanol. The
basic argument stems from the mismatch between the observed and the calculated
methane production due to hydrogen oxidation. Calculated hydrogen consumption
(using apparent kinetic constants), at the low hydrogen partial pressures usually
observed in anaerobic digesters and sediments, is not sufficient to account for
the observed methane production rate. They concluded that another molecule
479
must supply the remaining transferred electrons. Thiele and Zeikus199 proposed
that this molecule is formate. Although formate diffuses more slowly than hy-
drogen, larger concentration gradients can be established and thus greater electron
transport rates between syntrophic microorganisms can be achieved.199 Most meth-
anogenic bacteria can use formate.200 Thus, formate could be a feasible inter-
mediate. Nonetheless, the discrepancy in the calculations of methane production
from hydrogen158-196-198 may be due to an artifact during the measurement of the
kinetic parameters of hydrogen uptake. As was stated earlier in this paper, half-
saturation constants for hydrogen uptake are likely to be high due to the presence
of mass transfer limitations during the experimental determination.111
Another important intermediate metabolite is acetate. Droste and Kennedy183
have analyzed the effects of aggregation upon the acetate uptake in methanogenic
systems. Based on coupled reaction and diffusion models, they compared the
theoretical rates of acetate uptake when acetate is transferred via the bulk liquid
and when it is transferred inside the aggregate and concluded that aggregation is
beneficial for acetate removal and consequently for the overall substrate removal
rate.
VIII. OVERVIEW AND SUMMARY
In this paper, we have reviewed the kinetics of anaerobic treatment by fol-

lowing the various subprocesses involved (see Figure 1). The analysis was based
on the nature (i.e., soluble, particulate) and chemical composition of the waste
as well as on the microbiology and biochemistry of this complex process. Avail-
able kinetic information was presented and, whenever feasible, was subprocess-
specific. In this section, an overview of anaerobic treatment kinetics is presented
by summarizing the information available and by identifying areas where further
research is needed.
In terms of the chemical composition of waste, most of the literature pertains
to carbohydrates. More research is needed for the delineation of the kinetics of
anaerobic degradation of fat and proteins. Hydrogen gas, carbon monoxide, and
formate are substances upon which research has recently focused. However, more
research is needed in the area of interspecies electron transfer.
Knowledge of the viable, as opposed to the total, microorganism concentration
in anaerobic treatment systems is of paramount importance. Ideally, the mea-
surement of kinetic rates should be based on the viable microbial population
density. However, this is a very difficult task, especially in systems dealing with
particulate organic substrates (e.g., primary and waste activated sludge). More
research in this area is also needed for a more accurate expression of the kinetics
of anaerobic treatment.
Most of the kinetic values reported in the literature have been estimated by
use of enrichment, mixed cultures, or pure cultures in laboratory-scale digesters.
Although these studies have yielded invaluable information as far as the bio-
chemistry and microbiology of anaerobic processes are concerned, they are limited
480
in terms of kinetics. Laboratory studies create artificial, albeit well-defined, en-
vironments which in many cases oversimplify the complexity of real-life systems.
Although a need definitely exists for pilot-scale experimentation for the more
accurate delineation of the kinetics of anaerobic processes, more research should
focus on real-life anaerobic treatment systems.
Our understanding of the transport and removal of paniculate matter in an-
aerobic biofilms is limited. The existing level of mathematical modeling of these
phenomena is not yet suited for engineering applications. External mass transport
rates in three-phase (i.e., gas-liquid-solid) packed- and fluidized-bed reactors are
strongly affected by gas flow rates. Consequently, scaling from laboratory to full-
scale reactors is still problematic. External mass transfer is not likely to be a rate-
limiting step for acetate and propionate removal under the operating conditions
commonly encountered in high rate anaerobic reactors. On the other hand, internal
mass transfer limitations for acetate and propionate removal are significant at
aggregate sizes >0.8 mm and 1.2 mm, respectively. However, both external and
internal mass transfer are likely to be rate-limiting for the removal of carbohydrates
and exogenously supplied hydrogen.
In general, our understanding of the interplay between product inhibition,
kinetics, energetics, and mass transfer limitations in methanogenic aggregates is
very limited. Additional research is necessary in the area of mass transfer in
anaerobic systems.
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Critical Reviews in Environmental Control

Kinetics of anaerobic treatment: A critical review

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Kinetics of Anaerobic Treatment: A

Anaerobic digestion is one of the oldest biological wastewater treatment

The objective of this paper is to review the available kinetic information in

1. Hydrolysis of complex, particulate organic materials

Anaerobic degradation of viable, biological solids (e.g., waste activated sludge,

AMINO ACIDS. SUGARS FATTY ACIDSw ALCOHOLS

FERMENTATION INTERMEDIARY PRODUCTS ANAEROBIC

Expressed as acetic acid.

A. Fundamental Relationships in Biological Growth Kinetics

The quantitative description of microbial growth kinetics has greatly advanced

If conditions are conducive to growth, the rate of microbial growth can be

where b = specific microorganism decay rate (T" 1 ).

2. Growth Yield and Substrate Utilization Rate

3. Effect of Substrate Concentration on the Microbial Growth Rate

where: (i, = maximum specific growth rate (T" 1 ); S = concentration of the

Equation 6 becomes zero-order for S >KS and first-order for S <KS.

By neglecting the microorganism decay coefficient, Contois derived the following

where um = p7(l + a) and B = a/Y(l + a).

where K = al(\ + a) = constant (dimensionless). Chen and Hashimoto35 used

B. Continuous Culture Models

By use of the above presented fundamental kinetic relationships and applying

where X = microorganism concentration (M L~ 3 ); 0 = hydraulic retention time

= ¿ § _ b. ~dS = ¿ * § . e = So(1 + b9e)

_ h. ZEE- ¿xs . o _ W + fee.)

^ BX+ S ' dt Y(BX + S)'

Chen and Hashimoto

For a CSTR at steady-state, the following expression holds true:

0 r = (YU - fe)-1 (12)

Combining Equations 6 and 12 yields:

For cases where So > Ks, then:

By using Ks = a So, the above equation reduces to the equation of effluent

C. Kinetics of Hydrolysis of Particulate Matter

where Fo = concentration of degradable, particulate organic matter at t = 0 (M

where Fo, F = influent and effluent concentration of degradable, particulate

D. The Rate-Limiting Step Approach

As previously described, the anaerobic treatment of complex organics is a

(Fo, F, and kh as previously defined). By definition, process failure will occur

HYDROLYSIS CONSTANT (kh , d"1)

III. STOICHIOMETRY AND BIOENERGETICS

The chemical composition of the organic matter subjected to anaerobic diges-

CJIJ)Jfe+-(2n + c - b - ^ - ^JH2O -> j CH4

a. Carbohydrates (e = 0.504; s = 0.496)

SOLIDS RETENTION TIME (Days)

SOLIDS RETENTION TIME (Days)

FIGURE 3. Effect of solids retention time on the observed yield

b. Protein (e = 0.710; s = 0.290)

C 4 H 6 0N + 1.478H2O + O.lMHCOf = 0.247C5H7O2N + O.3OOCH3COO-

Carbohydrates 1.02 0.350 15.9 0.18

Hydrogen gas 22.55 0.030 29.2a 1.15

True yield coefficient.

c. Fat (e = 0.948; s = 0.052)

C15H3,COO- + 0.235NH4+ + 6.822HCO3~ = 0.235C5H7O2N

d. Butyrate (e = 0.918; s = 0.082)

CH3CH2CH2COO- + 0.082NH4+ + 0.918HCO3" = 0.082C5H7O2N

e. Propionate (e ~ 0.949; s = 0.051)

CH3CH2COO- + 0.036NH4+ + 0.407H2O = 0.036C5H7O2N