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Biochemical Engineering Journal 37 (2007) 219–225

Kinetic analysis of anaerobic digestion of cattailby rumen microbes in a modified UASB reactor

Zhen-Hu Hu a,b, Han-Qing Yu a,∗, Zheng-Bo Yue a, Hideki Harada c, Yu-You Li c

a Lab of Environmental Engineering, School of Chemistry, University of Science & Technology of China, Hefei 230026, Chinab School of Civil Engineering, Hefei University of Technology, Hefei 230092, China

c Department of Civil Engineering, Tohoku University, Sendai, 980-8579, Japan

Received 30 November 2006; received in revised form 25 April 2007; accepted 25 April 2007

bstract

The kinetics of anaerobic digestion of cattail by rumen microbes in a modified upflow anaerobic sludge blanket (UASB) reactor was systematicallynalyzed in this study. The Monod and first-order equations were combined to develop kinetic models to describe the substrate degradation,icrobial growth and product formation. At an influent cattail concentration (volatile solids) of 12.1 g/L, hydraulic retention time of 0.75 day and

olids retention time of 1.0–4.0 days, the concentration of reducing sugars was negligible, compared to the hydrolyzed cattail, indicating that the

ignocellulosic hydrolysis was the rate-limiting step in the fermentation. The rate constant (k) of cattail degradation, true microbial growth yieldYEG) and the maintenance coefficient (m) were calculated as 1.50 day−1, 0.11 g/g and 0.103 g/(g h), respectively. The measured and estimatedubstrate degradation, microbial growth and product formation were in good agreement.

2007 Elsevier B.V. All rights reserved.

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eywords: Anaerobic digestion; Cattail; Kinetics; Lignocellulose; Rumen micr

. Introduction

Cattail is an aquatic plant mainly composed of lignocellu-ose and widely used in constructed wetlands for the treatmentf wastewater and contaminated soil [1–4]. Anaerobic conver-ion of cattail has a great potential for disposal of the biomassroduced and more intensive application in pollution abatement5]. In addition, it can also be used for the generation of a vari-ty of fermentation products, which can be further recovereds energy resources [6]. Despite its significance, limited effortsave been made on the anaerobic digestion of cattail.

Because of the low cellulolytic activity and slow specificrowth rates of the anaerobic microorganisms involved, thenaerobic conversion efficiencies of lignocellulosic materialsre usually very low in conventional bioreactors [7]. However,his could be improved by enriching microorganisms with high

ellulase activities [8]. Our previous investigations and othertudies, all showed that the application of rumen microbes couldnhance this process [8,9]. Rumen microbes are composed of

∗ Corresponding author. Fax: +86 551 3601592.E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).

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369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2007.04.013

Upflow anaerobic sludge blanket (UASB)

hree types of microorganisms, bacteria, fungi and protozoa [10].n the rumen, bacterial numbers are very high and bacteria playdominant role in the fermentation of fibers [10]. But for sub-

trates with high fiber content, the rumen microbes appear toave large fungi population [11]. For lignocellulosic anaerobicigestion, the bacteria and fungi first attach to the surface ofignocellulosic materials, and then secrete enzyme to digest theomponents [11]. However, in the conversional artificial rumeneactors, the rumen microbes are usually in low concentrations12]. In this study, an upflow anaerobic sludge blanked (UASB)eactor was modified to increase the microbial concentration formproving the anaerobic digestion of cattail.

The anaerobic conversion of lignocellulosic material iscomplex process. Several different groups of bacteria are

nvolved and various end products are formed in the process12]. Generally, lignocellulosic wastes are first hydrolyzed byellulolytic microorganisms into low-molecular weight carbo-ydrates, which are then fermented into volatile fatty acidsVFA) by fermentative bacteria. The VFA are further converted

y acetogens into acetate and H2/CO2, both of which are lastlyormed to methane by methanogens [13]. This process has alsoeen widely used to treat a variety of municipal, industrial andnimal wastes to improve energy recovery [6]. The understand-

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ppsatwtfinptwapocontents were continuously stirred in order to keep the suspen-sion of particulate cattail. The influent cattail concentration was12.1 g VS/L. The reactor was fed once per 0.5 h and the residuewas removed once per 2 h. All experiments continued another

20 Z.-H. Hu et al. / Biochemical En

ng of the process kinetics is essential for the rational design andperation of anaerobic waste treatment systems in terms of goodystem stability and effluent quality.

Despite the significance of the anaerobic digestion of ligno-ellulosic wastes, only a limited number of studies have beenarried out on the kinetics of lignocellulosic digestion by anaer-bic bacteria. The Monod equation has been successfully usedn studying the kinetics of cultures utilizing simple substrates14]. However, for complex organic materials, the Monod modelould not well predict the reduction in volatile solids (VS) duringnaerobic fermentation of municipal refuse [15]. A first-ordereaction has been employed to describe the VS reduction of com-lex organic wastes [16]. In a previous study, Pavlostathis et al.12] have developed a kinetic model for describing the celluloseermentation by Ruminococcus albus in a continuously stirredank reactor (CSTR). In their work, hydraulic retention timeHRT) was equal to solids retention time (SRT) in a CSTR, andsimple substrate, i.e., cellulose, was used as the sole carbon andnergy source. In addition, a pure culture, R. albus, was used.

In our previous study, modified UASB reactor has showed aood potential to increase microbial concentration in the reactor,esulting in enhanced conversion efficiency for lignocellulose-ich substrate [9]. In this reactor, HRT was uncoupled with SRT.owever, without kinetic analysis on this bioreactor, and itsaste stabilization and products formation rates could not beredicted.

In this paper, the Monod and first-order equations were com-ined to describe the rates of substrate degradation, microbialrowth and product formation for the anaerobic digestion ofattail, a lignocellulose-rich substrate, by rumen microbes inhe modified UASB reactor. The results from this study werexpected to be helpful for designing and operating modifiedASB reactors for the high-efficiency anaerobic digestion of

ignocellulosic materials.

. Materials and methods

.1. Substrate, seed and media

Cattail with moisture content 60–70% was obtained from

river near the campus, Hefei, China. The sun-dried cattailas milled to 40-mesh powder, and was then used as the sub-

trate. The composition of cattail is shown in Table 1. Cattailontained 90.2% total solids, in which 64.1% was neutral deter-

able 1omposition of cattail

tem Percentage

otal solids (TS) (%) 90.2 ± 1.3olatile solids (VS) (% TS) 91.2 ± 2.3sh (% TS) 8.8 ± 0.3eutral detergent fiber (% TS) 64.1 ± 6.2cid detergent fiber (% TS) 41.5 ± 5.3emicellulose (% TS) 22.6 ± 2.5ellulose (% TS) 20.8 ± 2.8ignin (% TS) 10.5 ± 1.4

ote: Standard deviations were calculated with three measurements.

F((b(

ing Journal 37 (2007) 219–225

ent fiber. Cellulose, hemicellulose, lignin and ash accountedor 20.8, 22.6, 10.5 and 8.8% of total solids, respectively.

Rumen fluid, obtained from a fistulated goat, was used as seedicroorganisms. The media consisted of following ingredients

in g/L): NaHCO3 8; KH2PO4 1; K2HPO4 3; CaCl2·2H2O 0.03;gCl2·6H2O 0.08; NH4Cl 0.18 and 1 mL modified Pfennigetal solution, which had the following composition (in g/L):nSO4·7H2O 0.1; MnCl2·4H2O 0.03; H3BO3 0.3; CoCl2·6H2O.2; CuCl2·2H2O 0.01; NiCl2·6H2O 0.02; NaMoO4·2H2O 0.03;eCl2·4H2O 1.5.

.2. Reactor and operation

Fig. 1 illustrates the schematic diagram of the modifiedlexiglass-made UASB reactor, which consisted of a reactionortion (2.5 L and 12.0 cm in inner diameter) and a gas–liquideparator portion (2.6 L). Comparing with conventional UASB,mechanical stirrer with a diameter of 10.0 cm was fixed in

he reaction portion to improve the mixing of solid substrate,hich was placed 4 cm above the influent port and 3 cm below

he residue effluent port. The reaction volume of 2.5 L was usedor the calculation of volumetric loading and HRT/SRT. Rumens generally maintained at 39.5 ◦C and neutral pH condition inatural systems. Therefore, this UASB was operated at tem-erature 39 ± 1 ◦C with heating bands and pH at 6.8 ± 0.2 inhe reaction portion. The HRT was kept constant at 0.75 dayith various SRTs from 1.0 to 4.0 days. This was achieved by

djusting the volume of removed residue by using a peristalticump. The reactor content was mixed every 5 min for a periodf 1 min at an agitation rate of 80 rpm. The nutrient reservoir

ig. 1. Schematic diagram of the modified UASB reactor. (1) Sludge blanket;2) mechanical stirrer; (3) temperature probe; (4) waste tube; (5) effluent tube;6) baffle; (7) peristaltic pump; (8) magnetic stirrer; (9) feed reservoir; (10) gasalloon; (11) electromotor; (12) gas collection tube; (13) liquid effluent tube;14) computer.

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Z.-H. Hu et al. / Biochemical En

–3 weeks after the steady state was reached. The steady stateas defined as the fluctuation of VFA concentration in the reac-

or and chemical oxygen demand (COD) in the effluent less than0% on a daily basis, which generally needed 3–4 weeks afterrevious operation conditions were changed.

.3. Analysis

Liquor samples taken from the reactor were centrifuged at000 g for 15 min, and the supernatant was filtered through0.45-�m cellulosic membrane for the analysis of VFA and

educing sugars. The VFA was determined using a gas chromato-raph (GC-6890N, Agilent Inc., USA) equipped with a flameonization detector and a 30 m × 0.25 mm × 0.25 �m fused-ilica capillary column (DB-FFAP). The temperatures of thenjector and detector were 250 and 300 ◦C, respectively. Theven temperature was initially at 70 ◦C for 3 min followed by aamp of 20 ◦C/min for 5.5 min and held at the final temperaturef 180 ◦C for 3 min. Nitrogen was used as the carrier gas with aow rate of 2.6 mL/min. The biogas produced was recorded dailysing a gas meter and was analyzed by an another gas chromato-raph (SP-6800A, Lunan Co., China) equipped with a thermalonductivity detector and a 1.5 m stainless-steel column packedith 5 A molecular sieve. The temperatures of injector, detector

nd column were kept at 100, 105 and 60 ◦C, respectively. Argonas used as carrier gas at a flow rate of 30 mL/min. Neutral deter-ent fiber, acid detergent fiber, cellulose, hemicellulose, ligninnd ash contents of particulate cattail were measured accord-ng to Goering and van Soest [17]. Total solids, VS and CODere analyzed according to the standard methods [18]. Reducing

ugars were determined using the anthrone method [19] with glu-ose as the standard. Cell dry weight was estimated by measuringicrobial protein as described by Pavlostathis et al. [12].

. Results and discussion

.1. Kinetic equations

For a completely mixed UASB reactor under steady stateonditions, the disappearance of cattail in the UASB reactorollowed the first-order kinetics with respect to the concentrationf particulate cattail [12]:

dC

dt= −kC (1)

here C is the biodegradable VS concentration of particulateattail and k is the rate constant for cattail reduction.

For organic wastes like agricultural and animal wastes, a por-ion of VS is not available to the microbes as substrate [9]. Thushe VS in this study was expressed as the portion which coulde utilized by the rumen microbes.

The relationship between the bacterial growth rate and con-entration of the growth-limiting substrate is usually expressed

y Monod equation:

= μMS

Ks + S(2) i

t

ing Journal 37 (2007) 219–225 221

here μ is the specific growth rate (day−1), μM the maximumpecific growth rate, S the substrate concentration (g VS/L) ands is the half-saturation constant. In the present analysis, S repre-

ents soluble sugar concentration since this is the actual substratetilized by the rumen microbes.

Microbial growth yield (Y) is defined as bacterial mass pro-uced per mass of substrate utilized as shown in the followingquation:

= −dX

dS(3)

here X is the microbial biomass concentration. Therefore, theubstrate utilization rate can be derived from Eq. (3):

dS

dt= − μMSX

Y (Ks + S)(4)

In the anaerobic digestion of cattail, the consumed substrates composed of two parts: one for microbial growth, and anotheror the microbial maintenance and endogenous metabolism [12].hus:

dX

dt= (μ − α)X (5)

here α is the specific maintenance rate which accounts forhe microbial endogenous metabolism and maintenance require-

ents [12].

.2. Estimation of rate constant for substrate degradation

For a completely mixed reactor with uncoupled HRTsnd SRTs under steady state conditions, the disappearance ofiodegradable particulate cattail equal to QC0 − ZC, whichhould equal to the hydrolyzed cattail, VkC, based on Eq. (1).herefore, the following equation can be derived:

V dC

dt= QC0 − ZC − VkC = 0 (6)

here C0 is the influent biodegradable particulate cattail con-entration, C the biodegradable particulate cattail concentrationnder steady state conditions, Q the hydraulic flow rate (L/day),the solid flow rate (L/day) and V is the reactor reaction vol-

me. Thus, the particulate cattail concentration under steadytate conditions can be calculated using the following equation:

= C0RC

R(1 + RCk)(7)

here R is the HRT (=V/Q) and RC is the SRT (=V/Z). The reten-ion time is the inverse of dilution rate. The model shows that, atgiven R and RC, the biodegradable cattail concentration under

teady rate conditions depended on the influent biodegradableattail concentration and the rate constant k. Eq. (7) is linearizednto:

RCC0 = 1 + kR (8)

RC

C

The values of C0 and C can be estimated by subjecting thenfluent cattail and its effluent residues to long-term batch diges-ion, but this method is time-consuming. Since the true cattail

222 Z.-H. Hu et al. / Biochemical Engineer

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Fig. 2. Estimates of the rate constants for particulate cattail degradation.

egradation efficiency was equal to (QC0 − ZC)/QC0, the fol-owing equation can be derived:

1

1 − DE= RCC0

RC(9)

here DE is true cattail degradation efficiency. The DE can bealculated by the following equation:

E = DE′1 − f

(10)

here DE′ is the apparent cattail degradation efficiency, cal-ulated from the difference in the influent and effluent totalarticulate cattail concentrations and f is the refractory coef-cient, which is the ratio of non-biodegradable cattail portion to

he total cattail, and is obtained by performing long-term batchigestion of cattail.

In this study, the maximum cattail degradation efficiencybtained in the batch digestion was 75.9% [9], thus the refrac-ory coefficient was 0.241. The values of R, RC, DE′, DE and ofCC0/RC are listed in Table 2. The plot of RCC0/RC versus RC

esulted in a regression equation (Fig. 2):

= 1.01 + 1.50x (11)

The regression had a correlation efficient of 0.927 (R2), sug-esting the applicability of Eq. (11). From the regression line

lope, the k value was estimated as 1.50 day−1, which was higherhan that of 0.94 day−1 obtained in the batch digestion of corntover by rumen microbes [9] and that of 1.18 day−1 in the batchigestion of cellulose by R. albus [12]. The difference in rate u

able 2easured and estimated kinetic constants at various HRTs and SRTs

un Retentiontime (days)

Measured(g COD/L days)

Predicted(g COD/L days)

DE′ (%) DE

RC R Liquid Gas Total

4.0 0.75 7.2 3.2 10.4 11.1 65.2 853.0 0.75 6.2 3.7 9.9 10.9 64.2 842.5 0.75 5.4 3.5 8.9 9.9 58.4 762.0 0.75 4.6 2.7 7.3 8.7 51.2 671.0 0.75 4.4 1.6 6.0 6.2 38.5 50

otes: RC, solids retention time; R, hydraulic retention time; DE, true degradation effici, substrate concentration under steady state; X, microbial biomass concentration; U,

ing Journal 37 (2007) 219–225

onstants is likely attributed to the differences in substrate type.his result also shows that the continuous anaerobic digestionf cattail in the modified UASB reactor was feasible.

.3. Kinetics of microbial growth

For this UASB reactor, the biomass concentration in the efflu-nt under steady state conditions was very low and could begnored. Thus, the mass balance for the microbial biomass cane established as:

V dX

dt= QX0 − ZX + VμMSX

Y (Ks + S)− VaX = 0 (12)

Under steady state conditions, the following equation canlso be written:

(QC0 − ZC + QS0 − QS) = VuMSX

Ks + S(13)

Therefore, the microbial growth model for this UASB systemould be expressed by substituting Eq. (13) into Eq. (12):

= X0RC + Y (C0RC − RC + S0RC − SRC)

R(1 + αRC)(14)

here X0 and S0 are the influent microbial biomass and solubleugar concentrations, X and S are the corresponding concentra-ions under steady state conditions.

Since both concentrations of microbial biomass and solubleugar in the influent cattail were very low, they could be ignored.ence, Eq. (14) can be simplified into:

= Y [RC(C0 − S) − C]

R(1 + αRC)(15)

Defining U as specific substrate utilization rate (mass sub-trate utilized per mass biomass per day), thus:

= C0 − S

RX− C

RCX(16)

The linearization expression can be obtained by substitutingq. (16) into Eq. (15):

1

RC= YU − α (17)

Table 2 lists the microbial biomass concentrations and U val-es at various solid retention times. The quantity 1/RC was

(%) RCC0/RC X (g/L) 1/RC (day−1) U (g/(g days)) RS (g COD/L)

.9 7.09 2.02 0.25 5.22 0.15

.6 6.49 1.89 0.33 5.46 0.12

.9 4.34 1.41 0.40 6.74 0.12

.9 3.11 1.12 0.50 7.53 0.11

.7 2.03 0.53 1.00 11.78 0.09

ency; DE′, apparent degradation efficiency; C0, influent substrate concentration;specific substrate utilization rate; RS, reducing sugars.

Z.-H. Hu et al. / Biochemical Engineer

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lotted versus U, as shown in Fig. 3, resulting in followingquation:

= −0.32 + 0.11x (18)

From the values of the slope and intercept, the growth yieldnd specific maintenance rate were determined as 0.11 g/g and.32 day−1, respectively.

Energy consumed for microbial growth composed of twoarts: for microbial growth and maintenance requirement. Theaintenance coefficient (m) can be calculated using the follow-

ng equation [20]:

= a

YEG(19)

here YEG is the true growth yield, which is obtained whenubstrate energy is entirely utilized for growth without any uti-ization for maintenance [12]. YEG can be estimated by followingquation:

EG = cY

c − dY(20)

here Y is the true microbial yield based on total use of substrateor growth with zero maintenance requirement, c the carbonraction of the substrate and d is the carbon fraction of microbialiomass [12].

In the present study, cattail was a lignocellulosic material andainly composed of cellulose and hemicellulose and only par-

ially utilized by microbes. In our previous study, the maximumraction utilized was 75.9% [9]. The carbon fraction of cellulosend hemicellulose are 0.444 and 0.454, respectively, with anverage fraction of 0.45 [21]. Assuming that biodegradable cat-ail was completely composed of cellulose and hemicellulose,

he carbon fraction (c) in the cattail utilized by the microbes is.34 (0.45 × 0.759 = 0.34). Assuming that the microbial biomasss represented by the formula C5H7O2N and its fraction is 90%f the cell dry weight [12], the microbial biomass fraction ishus 0.48. The YEG is 0.13 g/g by substituting the values of c, dnd Y. Therefore, the maintenance coefficient was estimated as.103 g/(g h).

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ing Journal 37 (2007) 219–225 223

.4. Kinetics of substrate utilization

Soluble sugars converted from cattail were a substrate foricrobes and its mass balance composed of four parts: flowing inith influent substrate, producing in the reactor, flowing out with

ffluent residue and utilizing by microbes. Thus, under steadytate conditions, the following equation can be written:

V dS

dt= QS0 − QS + VkC − VμMSX

Y (Ks + S)= 0 (21)

Y [Q(S0 − S) + VkC] = VaX + ZX − QX0 (22)

here S0 is the influent soluble sugars concentration and S is theoluble sugar concentration under steady state conditions. Theinetic model of substrate utilization can be derived throughubstituting Eq. (22) into Eq. (21):

= Ks[(−X0/R) + X(1/RC + α)]

(X0/R) + X(μM − α − (1/RC))(23)

Eq. (24) gave the simplified form of Eq. (23) when X0 is equalo zero:

= Ks((1/RC) + α)

μM − α − (1/RC)(24)

The constants μM and Ks can be theoretically estimatedhrough the linearization of Eq. (24). However, such a plot ofata resulted in a negative correlation, because the observed sol-ble sugar concentrations showed a slight increase (Table 2),ather than a decrease, with increasing SRT. In fact, the solu-le sugar concentrations were very low as compared with theoncentration of the hydrolyzed particulate cattail. Therefore,rom a practical point of view, soluble sugar concentrations wereegligible. A similar problem was also encountered in the fer-entation of insoluble cellulose by pure rumen bacterium R.

lbus [12].

.5. Kinetics of product formation

The main products converted from cattail by rumen microbesn this UASB reactor were VFA, such as acetate, propionate,utyrate, valurate and their isomers, methane and carbon diox-de. For the calculation convenience of product formationinetics, the COD concentration was used as an indicator ofarticulate cattail, soluble sugars and product concentrations.he biodegradable COD concentration of particulate cattailould not be measured directly because of the existence of non-iodegradable organic fraction of cattail. Their COD values werealculated by the biodegradable fraction, assuming this fractions completely composed of cellulose and hemicellulose. TheOD values of VFA and methane were calculated based on

heir concentration, volume and theoretical conversion factor9], and the sum of both the items was the COD value of theain products, called measured COD, as presented in Table 2.

orrespondingly, the product COD value estimated from theinetics model was called predicted COD.

Mass conservation reasoning was used to perform the massalance of product, the substrate utilized either incorporated into

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24 Z.-H. Hu et al. / Biochemical En

ellular material or converted to products. Therefore, the amountf products should always be equal to the difference between theubstrate utilized and the net cellular biomass produced. Thushe following equation can be obtained:

V dP

dt= QP0 − QP + VμMSX

Y (Ks + S)

−VβX

[μMS

Y (Ks + S)− α

]= 0 (25)

here P0 is the influent products concentration, P the productsoncentration under steady state conditions, β is a factor used toonvert biomass to equivalent COD units (i.e., β = 1.28 g CODquivalent g of microbial biomass/dry weight). The net micro-ial growth is always equal to the loss from the effluent underteady state conditions. Thus, the new kinetic model for productormation can be written as:

R(C + βX)

= P0 + C0 + S0 − S + βX0 −RC

(26)

The specific product output rate (RP) is defined as the massf products generated per reactor volume per day (QP/V). Since

ig. 4. Model prediction of: (a) degradation efficiency; (b) microbial biomass;c) specific product formation rate.

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ing Journal 37 (2007) 219–225

0, S0 and X0 all equal to zero, substitution of RP into Eq. (26)ields the following equation:

P = C0 − S

R− C + βX

RC(27)

.6. Model verification

The effluent quality, treatment efficiency, volumetric sub-trate reduction rate and volumetric product output rate are theain factors in design and operation of a biological treatment

ystem [15]. With Eqs. (8), (15) and (27) developed from theonod and first-order reaction models, as well as the kinetic

onstants estimated from the data in Table 2, the apparent cattailegradation efficiency, microbial biomass and specific productutput rate in this modified UASB reactor could be calculated asfunction of SRT. Fig. 4a and b shows that, at any given SRT andRT as well as particulate cattail concentrations, the measured

nd estimated degradation of cattail and microbial growth fittedell. However, the formation rate of products estimated from theodel was greater than the measured value by 10% (Fig. 4c).

. Conclusions

A combination of the Monod equation and first-order equa-ion were used to establish models for describing the degradationf cattail, growth of microbes and formation of products inmodified UASB reactor. At an influent cattail concentration

f 12.1 g VS/L, hydraulic retention time of 0.75 day and solidsetention time of 1.0–4.0 days, the production of reducing sug-rs in the fermentation was negligible, compared to that of theydrolyzed cattail. The rate constant (k) of anaerobic cattailegradation was 1.50 day−1, the true microbial growth yieldYEG) was 0.11 g/g and the maintenance coefficient (m) was.103 g/(g h). Generally, the models were able to predict theates of cattail degradation, microbial growth well and productormation in this modified UASB reactor fed with a complexignocellulose-rich substrate.

cknowledgements

The authors wish to thank the Natural Science Foundation ofhina (Grant No. 20377037), NSFC-JST Joint Project (Granto. 20610002) and National Basic Research Program of China

Grant No. 2004CB719703) for the partial financial support ofhis study.

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