Modeling of fermentative hydrogen production from the bacterium Ruminococcus albus: Definition of metabolism and kinetics during growth on glucose

international journal of hydrogen energy 34 (2009) 3697–3709

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Modeling of fermentative hydrogen production from the bacterium Ruminococcus albus: Definition of metabolism and kinetics during growth on glucose I. Ntaikoua,b,*, H.N. Gavalaa,b,1, G. Lyberatosa,b a

Department of Chemical Engineering, University of Patras, Karatheodori 1 Street, 26500 Patras, Greece Institute of Chemical Engineering and High Temperature Chemical Processes, 26504 Patras, Greece

b

article info

abstract

Article history:

The aim of the present study was to describe the fermentative pathway of Ruminococcus

Received 29 January 2009

albus during hydrogen production from glucose by a quantitative kinetic model, taking into

Received in revised form

account the interactions among the metabolic products during their generation. Proper

21 February 2009

mathematical expressions were developed in order to adequately describe the microbial

Accepted 22 February 2009

growth and metabolism of R. albus. For the estimation of kinetics constants of the process,

Available online 25 March 2009

the experimental data from batch experiments were simulated using a simplified and modified version of Anaerobic Digestion Model 1 on Aquasim as a modeling platform.

Keywords:

Subsequently the accuracy of the model was verified by simulating the performance of

Biohydrogen

a CSTR in four different hydraulic retention times. Batch experiments with different initial

Ruminococcus albus

substrate concentrations and different initial hydrogen partial pressures were carried out

Metabolism

in order to calculate the growth kinetics of the microorganism and investigate the effect of

Kinetics

hydrogen partial pressure to the production of metabolites. Microbial growth was

ADM-1

described using Monod kinetics, taking into account the inhibition at lower pH values as well as the substrate inhibition, and the metabolites’ profile was described using suitable kinetic expressions. Acetate and ethanol production were assumed to occur simultaneously, by direct sugar consumption and the H2 final yield was reversely connected to the accumulation of ethanol. Formate was considered to be produced by direct sugar consumption, and subsequently to break down to H2 and CO2. The degradation rate of formate, and consequently hydrogen production were shown to be influenced by hydrogen partial pressure. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

A constantly increasing interest has evolved during the previous decades in sustainable energy. The severe and rapid global

climate changes along with the fear of energy supply shortage have created a large solicitude about the potential benefits of a hydrogen economy based on renewable energy sources [1–3]. This is due to the fact that hydrogen is a clean and renewable

* Corresponding author. Department of Chemical Engineering, University of Patras, Karatheodori 1 Street, 26500 Patras, Greece. Tel.: þ30 2610 996 577; fax: þ30 2610 993 070. E-mail address: [email protected] (I. Ntaikou). 1 Present address: Copenhagen Institute of Technology (Aalborg University Copenhagen), Department of Biotechnology, Chemistry and Environmental Engineering, Lautrupvang 15, DK 2750 Ballerup, Denmark. 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.02.057

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international journal of hydrogen energy 34 (2009) 3697–3709

energy carrier, possessing a high energy yield and not contributing to the greenhouse effect [4]. Hydrogen can be produced in a number of ways, such as through fossil fuels processing or by electrolysis using solar power, processes that could be prohibitively expensive [5]. On the other hand, the production of hydrogen via biological methods (using microorganisms) is gaining increasing attention as a sustainable alternative to the conventional methods for H2 production [6]. Biological production of hydrogen is usually divided into two main categories, according to the microorganisms that conduct it: photosynthetic and fermentative [7]. Fermentative bacteria which have the potential of converting biomass (plants, wastes) to valuable liquid or gaseous substances are used in the second case, during which carbohydrates are the main substrate for hydrogen generation [8–11]. The theoretical maximum yield of hydrogen generated via dark fermentation from sugars is reported to be 4 mol of hydrogen per mole of glucose if all the substrates would be converted to acetic acid [12]. Ruminococcus albus is a non spore-forming, obligatory anaerobic, coccoid bacterium [13,14] the natural habitat of which is the first stomach (rumen) of the ruminants. The ruminal ecosystem is constituted by bacteria, protozoans and fungi that interact either synergistically or antagonistically [15,16] providing nutrients to their host by breaking down the complex carbohydrates the latter consumes in the form of plant biomass, via fibrolytic enzymes [13,15,17,18]. R. albus together with Ruminococcus flavefaciens are reported to be the predominant species of the rumen, where they form populations that reach up to 59.8% of the total rumen microfauna [19]. These bacteria can ferment soluble carbohydrates such as cellobiose, glucose, xylose and arabinose, producing short chain acids, alcohols and hydrogen [20–22]. The main scientific interest on R. albus has so far focused on its fibrolytic capacity, since it produces all the hydrolytic enzymes which are necessary for breaking down two of the three polymers that form plant biomass, i.e. cellulose [23] and hemicellulose [24,25]. These studies refer to the identification of the genes, which are responsible for the production of the abovementioned enzymes [26–30], the enzymatic activity [14,31–33], and the cohesion mechanism of the fibers to be hydrolysed [34–36]. Possessing this hydrolytic possibility, R. albus is a very promising candidate for fermentative hydrogen production from biomass rich in cellulose/hemicellulose. It has already been shown that R. albus grows successfully on cellobiose, xylose and arabinose, which are the main products of cellulose and hemicellulose hydrolyses, leading to very promising hydrogen yields of 2–2.8 mol hydrogen per mole of glucose equivalents consumed. The hydrogen production from sweet sorghum biomass, an annual C4 energy plant, using R. albus has also been investigated, resulting in approximately 60 l of hydrogen per kg of wet sorghum biomass [22]. In the study of Ntaikou et al. [22] it has been proposed that R. albus metabolizes glucose via the metabolic pathway shown in Fig. 1. Regarding formic acid formation and degradation, it has been shown that R. albus follows the Enterobacteriaceae metabolic path under anaerobic conditions, according to which pyruvate is converted to acetylCoA and formate through pyruvate formate lyase. Subsequently, formate breaks down via formate hydrogen lyase to hydrogen and

a glucose 2a ADP + 2aPi 2a ATP

2a NAD+ 2a NADH +2aH+

2a pyruvate 2a CoA

2a acetylCoA

2a formate

ax NADH +ax H+

a(2-x) Pi a(2-x) CoA 2a H 2 ax acetaldehyde a(2-x) acety1- P

ax CoA

ax NADH +ax H+ ax ethanol

ax NAD+

ax NAD+

2a CO2

a(2-x) ADP a(2-x) ATP

a(2-x) acetate

Fig. 1 – Assumed metabolic path of glucose consumption by Ruminococcus albus.

carbon dioxide [37] according to Equation (1), whereas ethanol and acetate are produced from acetylCoA (Fig. 1). HCOOH / CO2 þ H2

(1)

This is in agreement with enzymatic studies on R. albus [38] that indicated the presence of enzymes, which lead to the Escherichia coli-type production of hydrogen and formic acid. Thus, formic acid is an important intermediate of R. albus metabolism and its production and degradation kinetics are among the key factors for efficient hydrogen production. However, little is known about the factors affecting the distribution of metabolic products and formic acid degradation to hydrogen and carbon dioxide. It has already been reported that the amount of hydrogen and in particular its partial pressure in continuous cultures of R. albus may influence the yields of the main metabolic products i.e. acetate and ethanol [39]. However, no kinetic model has been proposed so far for the description of the metabolism of the microorganism and the prediction of hydrogen production. The aim of the present study was to investigate the metabolism kinetics of R. albus during glucose consumption, taking into account the effect of hydrogen partial pressure ðPH2 Þ on the fate of the metabolites, using Anaerobic Digestion Model 1 on Aquasim as a modeling platform. The influence of pH and PH2 on formic acid degradation has been investigated and incorporated in the model as well. Batch experiments were used to develop the model, while experimental results from CSTR experiments at different hydraulic retention times were used to verify the developed model.

2.

Materials and methods

2.1.

Organism, medium and growth conditions

R. albus, strain DSMZ 20455 [40] was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen

international journal of hydrogen energy 34 (2009) 3697–3709

(DSMZ) and was maintained in a modified DSMZ 453 medium that was prepared as reported previously by Ntaikou et al. [22]. Stock cultures were stored at 22  C in 20% glycerol and inoculation cultures were transferred twice before use. All cultures were grown under a CO2/N2 (29:71, v/v) initial atmosphere, at 37  1  C and continuous stirring of 200  10 rpm and in all cases glucose was used as carbon source.

2.2.

Analytical methods

The measurement of hydrogen was carried out by a gas chromatograph (Varian Star 3600) equipped with a thermal conductivity detector and a packed column with nitrogen as carrier gas. Soluble fermentation products were measured by two different chromatographic methods. For the quantification of acetic acid and ethanol, acidified samples with H2SO4 (0.6% v/v) were analyzed on a gas chromatograph (Varian CP3800), equipped with a flame ionization detector and a capillary column. The detection limit values were 40–5870 mg/l for acetate and 50–4260 mg/l for ethanol. Formic acid was measured by ion chromatography (Dionex DX300) with an anionic column AS11 and conductivity detector CDM-3. The eluents used were NaOH 5 mM, NaOH 100 mM and purified water Milli-Q (Millipore), pressurized with He, and the liquid flow was kept constant at 1.5 ml/min. A gradient method was used starting with a concentration of 2.25 mM NaOH, until 65 mM NaOH. The detection limit values were 0.5–21 mg/l and the samples were diluted with purified water up to 1:100. Before analysis of organic products, the liquid samples were centrifuged at 10,000 rpm (15,652  g) for 10 min, and supernatants were passed through a membrane filter (0.2 mm pore size). For total and soluble (following centrifugation and filtration of the supernatant) carbohydrates’ determination, a colored sugar derivative was produced through the addition f L-tryptophan, sulphuric and boric acids [41,42], which was subsequently measured colorimetrically at 520 nm. The detection limits of the method were 5–150 mg/l. For biomass estimation, optical density was measured at 550 nm and subsequently biomass concentration was determined via the equation Cbiom ¼ (285.76  OD  3.212) mg/l, (R2 ¼ 0.999).

2.3.

Batch experiments

Three series of batch experiments were conducted, using serum vials of 161 ml total volume sealed with rubber stoppers and aluminium crimps. All cultures were performed in duplicates. The first series consisted of three batches with the same initial concentration of glucose (5 g/l) and different ratios of liquid to gas phase volumes, and aimed to the investigation of the effect of PH2 to the degradation of formate. In two of the batches PH2 increased due to the accumulation of the produced hydrogen in the gas phase of the sealed vials, whereas in the third one, PH2 was kept to zero by flashing away the produced gases with continuous gas flow of CO2/N2 (29:71 v/v). PH2 and formic acid concentration were followed versus time. The second series was conducted in order to estimate the growth kinetics of the microorganism on glucose. It consisted of four batches with different initial glucose concentrations, i.e. 4 g/l, 5 g/l, 8 g/l and 12 g/l and zero initial PH2 . Glucose and biomass concentrations and pH were

3699

followed versus time in order to determine the growth kinetics of he microorganism. The third batch series was conducted in order to estimate the kinetic constants and yields of the metabolites’ production. It consisted of five batch cultures with 5 g/l initial glucose concentration and different initial hydrogen partial pressures. In four of them the produced hydrogen accumulated and the initial PH2 had the values of 0.0 atm, 0.3 atm, 0.5 atm and 0.6 atm, whereas the fifth was continuously supplied with a mixture of CO2/N2 (29:71 v/v) at a rate of 25 ml per min in order to keep PH2 at zero, whereas the partial pressure of CO2 was kept at 0.29 atm. Glucose, biomass and metabolic products’ (acetic and formic acid and ethanol) concentrations, PH2 and pH were followed versus time.

2.4.

Continuous experiments

All continuous experiments were performed in a 2.5 l Virtis Omni-culture bench-top chemostat with working volume of 1320 ml and headspace of 1630 ml. The medium supply and the operation conditions were as described previously by Ntaikou et al. [22]. The operating hydraulic retention times (HRTs) tested were 48, 42, 36 and 24 h. Glucose, biomass and metabolic products’ (acetic and formic acid and ethanol) concentrations and pH were measured at each steady state and compared with the predicted ones in order to validate the kinetic model describing the growth of R. albus.

2.5.

Kinetic expressions and modeling

In order to describe substrate consumption a modified equation of Monod kinetics (Equation (2)) was used. The modified Monod equation included an inhibition factor (IpH) allowing for pH inhibition when low-pH inhibition occurred, and an inhibition factor (IS) allowing for substrate inhibition: dS S $X$IpH $IS ¼ km $ dt KS þ S

(2)

where km is the maximum specific rate of substrate consumption, which is equal to the ratio of maximum specific growth mmax to R. albus biomass yield Yx/S, S is the substrate concentration, Ks is the saturation constant, X is the biomass concentration. The factor IpH is a function of pH as shown in Equation (3) [43]:

IpH ¼ exp  3$

2   pHmeas  pHUL pHUL  pHLL

(3)

where pHmeas is the measured pH value, pHUL is the pH value for which microbial growth is not inhibited and for R. albus is 7.0 and pHLL is the pH value which actually controls the inhibition function. The factor IS of Equation (2) is a function of S which describes non-competitive substrate inhibition according to Equation (4) [44]: IS ¼

KIS KIS þ S

(4)

where KIS is the value of substrate concentration when the substrate consumption rate is half of the maximum.

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international journal of hydrogen energy 34 (2009) 3697–3709

R. albus growth kinetics were described by Equation (5): dX S $X$IpH $IS $YX=S  kd $X ¼ km $ dt KS þ S

(5)

where kd is the decay constant and Yx/S is the microbial biomass yield. Based on Monod kinetics the production rates of acetate and ethanol, we described by Equation (6): dSP S ¼ YP=S $km $ $X$IpH $IS dt KS þ S

(6)

where YP/S and SP were the yield and concentration of each product respectively. Formate production was described by modified Monod kinetics, whereas its degradation was described by first order kinetics, depending on microbial biomass and PH2 as shown in Equations (7) and (8): dSF S ¼ YF=S $km $ $X$IpH $IS  KF $SF $X$IH2 dt KS þ S

Hydrogen partial pressure (atm)

a

(7)

1,2

0,8 0,6 0,4 0,2

0

5

10

15

20

25

30

35

time (h)

0,7 0,6

Formate (kg COD/m3)

Liquid:gas phase (v/v)

Initial PH2 (atm)

Final PH2 (atm)

Final formate concentration (kg COD/m3)

1:1 1:3 1:1

0  0.00 0  0.00 0

0.94  0.01 0.65  0.01 0

0.239  0.00 0.210 0.023

where YF/S and SF are the yield and concentration of formate respectively and KF is the formate degradation constant. IH2 ¼

1

(8)

P

H2 1 þ KIH

2

where PH2 is H2 partial pressure and KIH2 is the H2 partial pressure inhibition constant. The production rate of hydrogen generated by both glycolysis and formate degradation, was described by Equation (9): dSH2 S ¼ YH2 =S $km $ $X$IpH $IS þ KF $SF $X$IH2 dt KS þ S

1,0

0,0

b

Table 1 – Final formate concentrations in batch cultures with 5 g/l initial glucose concentration and different profiles of PH2 built up.

0,5 0,4 0,3

(9)

where YH2 =S is the hydrogen yield from glucose consumption. For the estimation of the COD equivalents for the biomass concentration the empirical formula C5H7O2N was used, which is commonly used for describing anaerobic bacteria and was also usedinprevious studiesfordescribingR. albusbiomass[23,45].The computer software ‘‘Aquasim 2.0’’ [46] was used for fitting of the equations to the experimental data and for simulating the chemostat behaviour. A simplified and modified version of mathematical model Anaerobic Digestion Model 1 [43] was developed and used as a platform for the simulations. The processes of hydrolysis, acetogenesis from volatile fatty acids and methanogenesis were excluded from the modified version used. At acidogenesis, the only substrate was glucose and apart from acetate production no other available equations for acids’ production were used. Formate and ethanol production from glucose were incorporated into the model, taking into account their pKa for pH estimations. Formate degradation was also incorporated. All organic substances and molecular hydrogen were described in terms of chemical oxygen demand (kg COD m3), whereas inorganic carbon, inorganic nitrogen and trace metals were described in terms of their molar concentrations (M).

0,2 0,1

Table 2 – Growth kinetic constants of Ruminococcus albus grown with glucose as carbon source.

0,0 0

5

10

15

20

25

30

35

time (h) Fig. 2 – Hydrogen partial pressure (a) and formate concentration (b) in batch cultures with 5 g/l initial glucose concentration and different profiles of PH2 built up. Symbols: C, experiment with accumulation of H2 and liquid phase:gas phase [ 1:1, :, experiment with accumulation of H2 and liquid phase:gas phase [ 1:3, 6, experiment with removal of the produced gases.

Kinetic constant kd mmax km Yx/S KS KIS pHLL

Values 1

1.02  0.056 (d ) 18.37  0.702 (d1) 99.60  6.086 (kg COD gl/kg CODX/d) 0.184  0.010 (kg COD X/kg COD gl) 0.698  0.021 (kg COD/m3) 25.34  1.583 (kg COD/m3) 5.13

0.042  0.002 (h1) 0.765  0.029 (h1) 5.234  0.320 (g gl/g X/h) 0.139  0.012 (g X/g gl) 0.654  0.039 (g/l) 23.76  1.484 (g/l)

international journal of hydrogen energy 34 (2009) 3697–3709

a

4,0

3.

Acetate, ethanol (kgCOD/m3)

3,5 3,0

2,0 1,5 1,0

0,0 0,0

0,2

0,4

0,6

0,8

1,0

1,2

time (d) 4,0

Acetate, ethanol (kgCOD/m3)

3,5 3,0 2,5 2,0

1,0

0,0 0,0

0,2

0,4

0,6

0,8

1,0

1,2

time (d)

Acetate, ethanol (kgCOD/m3)

2,5 2,0 1,5 1,0

0,0 0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,8

1,0

1,2

time (d) 4,0

Acetate, ethanol (kgCOD/m3)

3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 0,0

0,2

0,4

0,6

time (d) 4,0 3,5

Acetate, ethanol (kgCOD/m3)

In order to estimate the growth kinetics of the microorganism, the experimental data of microbial biomass and substrate concentrations and pH values from the first and third batch series were simulated using Equations (2)–(5). The estimated values are shown in Table 2.

3.3. Metabolic product generation mechanism and modeling

3,0

0,5

e

Growth kinetics

4,0 3,5

d

The experimental data of the first batch series indicated that PH2 has a severe effect on formate degradation in terms of both its degradation rate and the total amount that is degraded. As shown in Fig. 2 the degradation rate of formate seems to get slower when the built up rate of PH2 gets faster. As also shown in Table 1, the increased PH2 not only affects the degradation rate of formate but also the residual amount that is left after the end of the whole procedure. It is noticeable that when PH2 is kept to zero almost the whole amount of produced formate is degraded.

3.2.

1,5

0,5

c

Results

3.1. Effect of hydrogen partial pressure on formate degradation

2,5

0,5

b

3701

3,0

In order to estimate the kinetics constants and yields of metabolites’ generation, the experimental data of the third batch series were simulated using Equations (2)–(9) and the already estimated growth kinetic constants. The only metabolic products detected were acetic and formic acids, ethanol, carbon dioxide and hydrogen. The metabolic products’ generation mechanism that was used for the determination of the mathematical equations which would describe the generation of metabolites, was based on the metabolic path which is illustrated in Fig. 1. According to the proposed metabolic path all soluble products are generated simultaneously via glycolysis, whereas gas products are generated by two distinct paths i.e. glycolysis and degradation of formate. Subsequently, acetic acid and ethanol concentrations remain constant after the full uptake of glucose, formic acid concentration decreases and a delayed production of hydrogen and carbon dioxide is observed. Based on Monod kinetics the production rates of acetate and ethanol were described by Equation (6). The experimental data and the simulations are shown in Fig. 3, whereas the experimental measured yields and the yields as estimated by the model are shown in Tables 4 and 5 respectively. As

2,5 2,0 1,5 1,0 0,5 0,0 0,0

0,2

0,4

0,6

time (d)

0,8

1,0

1,2

Fig. 3 – Experimental data and theoretical profiles of acetate and ethanol concentrations during batch experiments with glucose initial concentrations of 5 g/l and different profiles of hydrogen partial pressure, PH2 initial 0 atm (a), PH2 initial 0 atm, PH2 initial 0.3 atm (b), PH2 initial 0.5 atm (c), PH2 initial 0.6 atm (d) and PH2 constant 0 atm (e). Symbols: 7 measured ethanol, ; measured acetate, d model prediction for ethanol, d model prediction for acetate.

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international journal of hydrogen energy 34 (2009) 3697–3709

Formate (kgCOD/m3)

a

0,6 0,5 0,4 0,3 0,2 0,1 0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,8

1,0

1,2

0,8

1,0

1,2

0,8

1,0

1,2

time (d) Formate (kgCOD/m3)

b

0,6 0,5 0,4 0,3 0,2 0,1 0,0

0,2

0,4

0,6

time (d) Formate (kgCOD/m3)

c

0,6 0,5 0,4 0,3 0,2 0,1 0,0

0,2

0,4

0,6

time (d) Formate (kgCOD/m3)

d

0,6 0,5 0,4 0,3 0,2 0,1 0,0

0,2

0,4

0,6

0,8

Formate (kgCOD/m3)

time (d)

e

0,7 0,6 0,5

indicated by these results, PH2 did not influence the acetic acid and ethanol yields at least at the range tested. Formate was considered to be produced by direct sugar consumption and subsequently to break down to hydrogen and carbon dioxide as described by Equation (7). Formate production was described by modified Monod kinetics, whereas its degradation was described by first order kinetics, depending on microbial biomass and the inhibition factor IH2 (Equation (8)). This factor was described as a function of the PH2 and the constant KIH2 as shown by Equation (9). The addition of the inhibition factor IH2 was proven to be necessary as shown from the experimental data of the first batch series, according to which the degradation rate of formate was strongly affected by the profile of PH2 built up. The values of the degradation constant of HCOOH, KF and the inhibition coefficient KIH2 , were estimated by simulating the experimental data of the third batch series using Equations (7) and (8). The yield of produced formate from glucose was estimated based on the metabolic path that is illustrated in Fig. 1, according to which, 2 mol of formate are produced per mole of glucose that is converted to metabolic products, and taking into account the amount of glucose that is converted to biomass which was 0.184 g COD X/g COD of glucose. Fig. 4 shows the experimental data and the prediction of the model for the concentration of formate. It is obvious that the theoretical curves adequately matched the experimental profiles. The estimated values from the parameter estimation of KF, and KIH2 , were 0.17 l g/X/h corresponding to 2.88 m3 kg COD/ X/d, and, 6.8 atm respectively. Since hydrogen is partially generated by formate degradation, its production rate and its final yield are expected to be affected by PH2 as well. The production rate of hydrogen, generated by both glycolysis and formate degradation, is described by Equation (9). As indicated by previous studies [47] hydrogen is generated during glycolysis by oxidation of the NADH that was produced, from which 2 mol of H2 are produced during regeneration of NADþ. As illustrated in Fig. 1, 2 mol of NADH are produced per mole of glucose that is converted to pyruvate, whereas 2 mol of NADH are uptaken for the production of 1 mol of ethanol. Consequently, since a part of the produced NADH is used during ethanol production, the final amount of hydrogen produced from glycolysis will decrease to an amount respective to the NADH consumption during ethanol formation. In order to simulate the experimental data of produced hydrogen from glycolysis, the dependence of the hydrogen production to ethanol production was expressed by connecting the yield of produced hydrogen with the yield of ethanol. Consequently, the molecular yield of hydrogen from glucose was estimated as 2(1  Yeth) per mole of glucose that is converted to metabolic products, with Yeth being the molecular ethanol yield.

0,4 0,3 0,2 0,1 0,0

0,2

0,4 0,6

0,8 1,0

time (d)

1,2 1,4 1,6

Fig. 4 – Experimental data and theoretical profiles of formate concentration during batch experiments with glucose initial concentrations of 5 g/l and different profiles of hydrogen partial pressure, PH2 initial 0 atm (a), PH2 initial 0 atm, PH2 initial 0.3 atm (b), PH2 initial 0.5 atm (c), PH2 initial 0.6 atm (d) and PH2 constant 0 atm (e). Symbols: B, measured formate, d, model prediction for formate.

international journal of hydrogen energy 34 (2009) 3697–3709

H2 production (kgCOD/m3)

a

1,0 0,8 0,6 0,4 0,2 0,0 0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,5

0,6

0,7

time (d)

H2 production (kgCOD/m3)

b

1,0 0,8 0,6 0,4 0,2 0,0 0,0

0,1

0,2

0,3

0,4

time (d)

H2 production (kgCOD/m3)

c

1,0 0,8 0,6

4.

0,2

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

time (d)

H2 production (kgCOD/m3)

1,0 0,8 0,6 0,4 0,2 0,0 0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

time (d)

H2 production (kgCOD/m3)

e

The experimental data and theoretical profiles of hydrogen production during the batch experiments with different PH2 are shown in Fig. 5. It is obvious, as indicated by Table 3, that PH2 not only has an impact on the hydrogen production rate, but also affects the final hydrogen yield. The latter becomes lower for higher PH2 and is similarly severely related with the residual amount of formate. The yields of the metabolic products and the sum of yields as estimated from the experimental data and by the model prediction are shown in Tables 4 and 5 respectively. It is obvious that the COD balance is closed in all cases for both measured and predicted values. The experimentally measured values of pH and the predictions of the model are shown in Fig. 6. For the simulation, the concentration of total anions and cations due to the inorganic elements of the medium and the concentrations of the organic acids that were produced by glucose metabolism were taken into account, as well as the acidicity constants of the acids at 25  C. It is obvious that the prediction of the model simulated the experimental data very successfully. In order to evaluate the accuracy of the proposed model, the performance of a continuous reactor at different HRTs was simulated, using the estimated values from the batch experiments. In Table 6, the experimental data and the prediction of the model are shown for all HRTs. It is obvious that the model predicted quite well all measured quantities. It should also be noted that the model successfully predicted the increase in biomass concentration for lower HRTs.

0,4

0,0

d

3703

1,0 0,8

Discussion

4.1. Effect of hydrogen partial pressure on formate degradation Hydrogen partial pressure ðPH2 Þ is reported to be one of the key factors that affects the fermentative production of hydrogen from various bacteria, leading to decreased yields when it builds up [48–51]. As assumed in the present study, hydrogen is partially produced by formate degradation and consequently it would be expected that PH2 would also have some impact to the degradation of formate. Indeed, as indicated by the experimental data of the first batch series of the present study, formate accumulation seems to be affected by PH2 . The final amount of formate detected in the culture with constant removal of hydrogen atmosphere was ten times less than the one detected in those cultures where PH2 was building up. The increase of observed amount of formate under high PH2 was also reported in studies with E. coli [52], according to which when PH2 was constantly kept at 1 atm the accumulated amount of formate was doubled in comparison to that

0,6 0,4 0,2 0,0 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

time (d)

Fig. 5 – Experimental data and theoretical profiles of hydrogen production during batch experiments with glucose initial concentrations of 5 g/l and different profiles of hydrogen partial pressure, PH2 initial 0 atm (a), PH2 initial 0 atm, PH2 initial 0.3 atm (b), PH2 initial 0.5 atm (c), PH2 initial 0.6 atm (d) and PH2 constant 0 atm (e). Symbols: >, measured hydrogen, d, model prediction for hydrogen.

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international journal of hydrogen energy 34 (2009) 3697–3709

Table 3 – Hydrogen yields as estimated from the experimental data and residual formate concentration in batch cultures with 5 g/l initial glucose concentration and different profiles of PH2 built up. Initial PH2 Final PH2 Molecular H2 H2 final yield Residual (atm) (atm) final yield (kg COD/ formate (mol/mol gl) kg COD gl) (kg COD/ m3) 0  0.00 0.3  0.01 0.5  0.01 0.6  0.00 0

0.98  0.01 1.06  0.03 1.08  0.00 1.09  0.00 0

2.01  0.01 1.77  0.01 1.57  0.00 1.47  0.00 2.23a

0.168  0.00 0.148  0.00 0.130  0.00 0.128  0.00 0.191a

0.245  0.00 0.263  0.00 0.270  0.00 0.272  0.00 0.025

a Theoretical values as predicted from the model.

accumulated when the PH2 is 0.05 atm. Based on these observations the degradation rate of formate was directly linked to PH2 in the gas phase of the cultures (Equations (9) and (10)).

4.2.

Growth kinetics

The estimated values of the growth kinetics as shown in Table 1, are slightly different from the ones estimated from a previous study of Ntaikou et al. [22] during which substrate inhibition was not taken into account. The inclusion of substrate inhibition factor in Equations (1) and (4), proved to be necessary since it was shown, that the previously estimated values of the growth kinetic parameters, could not simulate satisfactorily the experimental data from cultures with high substrate concentrations.

4.3. Metabolic product generation mechanism and modeling The proposed model of the present study was based on the assumption that R. albus catabolises glucose following a similar metabolic path to that used by Enterobacteriacea, as previously proposed by Ntaikou et al. [22]. According to this pathway, NADH is produced during glycolysis, pyruvate is

Table 4 – Experimentally measured yields and total experimentally estimated equilibrium in batch cultures with 5 g/l initial glucose concentration and different profiles of PH2 built up. Initial PH2 (atm)

0  0.00 0.3  0.01 0.5  0.01 0.6  0.00 0

Experimentally measured yields (mg COD/mg COD gl) Formate

0.049  0.00 0.052  0.00 0.053  0.00 0.054  0.00 0.004

Acetate

0.25  0.00 0.25  0.01 0.23  0.00 0.23  0.00 0.23

Ethanol

0.41  0.00 0.47  0.02 0.42  0.00 0.41  0.00 0.40

H2a

0.168  0.00 0.148  0.00 0.130  0.00 0.128  0.00 0.191b

Some of yields with biomass 1.01  0.01 1.01  0.03 1.01  0.01 1.01  0.01 1.01

a From both direct glucose consumption and via formate degradation. b Theoretical values as predicted from the model.

metabolized to phosphoacetylCoA and formate, in equal amounts, ethanol and acetate are derived from phosphoacetylCoA in ratios depending on the prevailing conditions and hydrogen is produced by both NADH oxidation and formate breakdown, with its final yield affected by the conditions [53– 56]. The kinetic equations that were developed based on the abovementioned assumptions about the metabolic path, led to a satisfactory description of the observed behaviour of R. albus. Growth kinetics of R. albus have also been estimated in previous studies for different substrates. Pavlostathis et al. [57] have studied the growth kinetics of R. albus with cellulose as substrate, estimating the main growth parameters as well as products’ generation in terms of specific product output rate. Growth kinetics of two different strains of R. albus with cellulose and cellobiose as carbon sources have also been investigated using 3-phenylpropanoic acid for stimulating the growth rate, by Stack and Cotta [58]. However the attempt to quantify the metabolism of R. albus using a kinetic model, in which both liquid and gaseous products would be taken into account, has not been made so far. On the other hand, the modification of ADM-1 so as to predict hydrogen production from carbohydrates has also been reported by Peiris et al. [59], leading to satisfactory simulations. The study of metabolism during hydrogen production and its quantitative description using a modified version of ADM-1 has also been conducted recently for four different clostridial species [60]. In this study of Lin et al. [60] the kinetic contacts were determined by simulating the data from batch experiments, without colligating the yields of different metabolic products in the equations used. The simulations were satisfactory in all cases, showing that the model was able to describe the trends of glucose consumption, biomass production, and fermentation product formation fairly well. Penumathsa et al. [61] further showed that ADM-1 is also adequate for the simulation of continuous biohydrogen production via mixed acidogenic cultures. As shown by the results of the present study the modified ADM1 has also proved to be adequate for simulating the metabolic processes that take place during fermentation of glucose by R. albus, predicting quite well both liquid and gaseous products’ generation, during both batch and continuous cultures. Moreover, the assumed dependence of

Table 5 – Estimated yields and total estimated equilibrium in batch cultures with 5 g/l initial glucose concentration and different profiles of PH2 built up, according to model predictions. Initial PH2 (atm)

Yields from model prediction (mg COD/mg COD gl) Formate Acetate

0  0.00 0.3  0.01 0.5  0.01 0.6  0.00 0

0.13 0.13 0.13 0.13 0.13

0.23  0.02 0.25  0.03 0.23  0.01 0.24  0.02 0.24  0.01

Ethanol

0.44  0.03 0.42  0.02 0.46  0.03 0.42  0.02 0.44  0.02

H2 (from Some of glucose) yields with biomass 0.08  0.01 0.08  0.01 0.07  0.01 0.08  0.01 0.08  0.01

1.07  0.09 1.07  0.09 1.08  0.08 1.05  0.07 1.07  0.08

international journal of hydrogen energy 34 (2009) 3697–3709

a

7,0 6,8

pH (log H+)

6,6 6,4 6,2 6,0 5,8 5,6 0,0

0,2

0,4

0,6

0,8

1,0

1,2

time (d)

b

2Fed(red) þ 2Hþ / 2Fed(ox) þ 2H2

pH (log H+)

6,6 6,4 6,2 6,0 5,8 5,6 0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,8

1,0

1,2

0,8

1,0

1,2

time (d) 7,0

pH (log H+)

6,8 6,6 6,4 6,2 6,0 5,8 5,6 0,0

0,2

0,4

0,6

time (d)

d

7,0

pH (log H+)

6,8 6,6 6,4 6,2 6,0 5,8 5,6 0,0

0,2

0,4

0,6

time (d)

e

7,0 6,8

pH (log H+)

metabolite yields on other metabolites proved to be appropriate, since in all cases the model simulated the experimental data very successfully. It was shown that PH2 does not affect the yields of acetate and ethanol, whereas it strongly affected both the degradation rate and the yield of formate and consequently the yield of hydrogen. The metabolic shift towards ethanol is reported to be NADH dependent [53]. During glycolysis, NADþ is reduced to NADH which has to be oxidised again so that the substrate consumption can be continued. This can be done either by ethanol production [53] or by molecular hydrogen formation from reduced ferredoxin [62], as shown in the following equation:

7,0 6,8

c

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(a)

6,6

(10)

The standard Gibbs free energy for Equation (10) at pH 7 is þ3.1 kJ/mol [59,63] and thus, for neutral pH this reaction is thermodynamically feasible until hydrogen partial pressure reaches 104.5 Pa (0.3 atm). The ability of R. albus to produce molecular hydrogen from NADH via ferredoxin was reported in previous studies [64], but the abovementioned limitation of partial pressure for the feasibility of this assumed reaction was not observed in our study. The generation of molecular hydrogen occurred at high hydrogen partial pressures, even in the range 0.6–1 atm. It has to be mentioned though that in all of the experiments described in the present study, the pH was acidic and not neutral. It is known that the Gibbs free energy is affected by pH values, and consequently the limit of 0.3 atm is certainly altered in the case of the experiments of the present study. The different pH values can also be responsible for the observed differences of the present study to that of Ianotti et al. [39], in which it has been reported that in continuous cocultures of R. albus and Vibrio succinogenes, the metabolism of R. albus shifted towards acetate instead of ethanol when low PH2 values occurred. In that study the pH value was kept constant at 6.8, whereas in this study the pH was allowed to drift between 6.8 and 5.9. It is reported that enterobacteria in order to avoid acidification of their cytoplasm when the extracellular pH drops, shift the metabolism of acetylCoA towards ethanol instead of acetate [54,65]. Consequently, since R. albus has similar metabolism to the enterobacteria, it can be postulated that the accumulation of ethanol that was observed at an equal ratio in all batch experiments of this study was mainly in response to the pH drop. The degradation mechanism of formate to molecular hydrogen and carbon dioxide also seems to be pH dependent and regulated. Doelle [66] reported that in E. coli cultures, formate broke down producing hydrogen when the pH of the medium was 6.2 whereas for pH 7.8, formate accumulated. Similar observations were also reported for the bacterium Enterobacter aerogenes [67]. According to Suppman and Sawers

6,4 6,2 6,0 5,8 5,6 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

time (d)

Fig. 6 – Experimental data and theoretical profiles of pH during batch experiments with glucose initial concentrations of 5 g/l and different profiles of hydrogen partial pressure, PH2 initial 0 atm (a), PH2 initial 0 atm, PH2 initial 0.3 atm (b), PH2 initial 0.5 atm (c), PH2 initial 0.6 atm (d) and PH2 constant 0 atm (e). Symbols: =, measured pH, d, model prediction for pH.

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international journal of hydrogen energy 34 (2009) 3697–3709

Table 6 – Experimental data and model prediction of continuous cultures with 5 g/l glucose, at different hydraulic retention times. HRT (h)

% Glucose uptake Biomass (kg COD/m3) pH Acetate (kg COD/m3) Formate (kg COD/m3) Ethanol (kg COD/m3) PH2 (atm)

48

42

36

Experimental

Model

Experimental

Model

Experimental

95.43  3.01 0.28  0.04 6.5  0.02 1.77  0.08 0.35  0.01 2.89  0.05 0.48  0.03

97.34 0.33 6.68 1.33 0.27 2.55 0.55

98.28  0.94 0.29  0.02 6.54  0.03 1.83  0.04 0.23  0.01 2.89  0.3 0.53  0.02

99.01 0.33 6.67 1.35 0.27 2.59 0.55

96.06  0.34 0.33  0.02 6.37  0.03 1.79  0.08 0.29  0.01 2.90  0.14 0.43  0.01

[68] the formate that was produced during the fermentation of glucose by E. coli was initially transferred out of the cytoplasm in order to avoid cytoplasmic acidification, where it accumulated to an amount up to 10 mM. When the pH of the culture dropped below 6.8, the accumulated formate was imported back to the cell where it was metabolized via the formate hydrogen lyase pathway. The expression of the genes which are responsible for encoding formate hydrogen lyase is regulated together with the gene which encodes the transportation protein of formate in and out of the cell. Since R. albus is reported to produce formate by pyruvate following the Enterobacteriaceae fermentation type [38], it can be assumed that it would follow the Enterobacteriaceae pathway for formate degradation, as well. However, the degradation rate of formate was not connected to the pH in any of the abovementioned studies. In the present study the pH was indirectly connected to formate degradation via the microbial biomass, the concentration of which is a function of pH. First order kinetics were used for describing the degradation of formate depending on the active biomass concentration, since formate hydrogen lyase, which is responsible for formate degradation to hydrogen and carbon dioxide, is an intracellular enzyme [69]. The value of the inhibition factor KIH2 , i.e the PH2 at which formate degradation would fully be inhibited, was 6.8 atm. Such a high PH2 could hardly be reached in reality, implying that the rate of degradation is affected by hydrogen accumulation in the gas phase to a much smaller degree than the residual amount of formate. De Corte et al. [52] claimed that in E. coli the low redox potential of the formate degradation reaction allows hydrogen to be released, irrespective of the PH2 in the gas phase, as long as it is kept below 1 atm. However, when the experimental data of the present study were simulated without using the PH2 factor in Equation (6), the model failed to reach a satisfactory fitting. It must be noted though that the model underestimated slightly the residual amount of formate in the cultures in which PH2 was building up, whereas it overestimated the residual amount of formate in the cultures that had zero PH2 . Respectively, hydrogen, which is partly generated from formate, was overestimated in the first case and underestimated in the second. It seems that there is some factor, irrelevant to the PH2 , which after a certain point does not allow the entrance of formate into the bacterial cell and its subsequent degradation towards hydrogen generation. This could be the increase of pH, since as already mentioned, the accumulated formate is imported back to the cell only when

24 Model 98.35 0.40 6.68 1.27 0.29 2.43 0.55

Experimental

Model

96.21  1.95 0.40  0.02 6.47  0.04 1.52  0.04 0.31  0.01 2.03  0.06 0.41  0.01

99.02 0.47 6.67 1.22 0.31 2.34 0.56

pH values are below 6.8 [68]. However, this was not the case in the present study. A possible limiting factor could be the carbon dioxide partial pressure ðPCO2 Þ in the gas phase, which is reported by previous studies to have a negative effect on the observed final hydrogen yield [70]. Indeed in the experiments with continuous removal of hydrogen via CO2:N2 atmosphere, the PCO2 was constantly equal to 0.3 atm, whereas in all the other cultures it is estimated that it reached up to 1.5 atm. The positive effect of CO2 removal on fermentative hydrogen production has also been reported in previous studies with the enterobacterium Enterobacter aerogenes [71]. Enhanced hydrogen production was attributed to the amount of residual NADH which was promoted by the removal of CO2 from culture. However, since no pH control was taking place during the experiments, the observed effect could also be connected to the smaller degree of pH decrease in the absence of dissolved CO2 in the medium. It is true indeed that pH is one of the main factors affecting hydrogen production [72] and that below certain values can be strongly inhibited [73,74]. In the case of the present study the presence of CO2 in the gas phase did not have a significant effect on the pH variation since buffer solution was used in all experiments aiming to maintain the pH drop into certain limits. However the hypothesis that PCO2 can affect hydrogen production from R. albus is under investigation and could possibly be clarified by further experiments using nitrogen for drifting away the produced gases by sparging. It is true indeed that sparging is strongly connected with increased hydrogen yields but in all cases this result is attributed to the efficient removal of the produced hydrogen in terms of reducement of hydrogen partial pressure and not with the removal of carbon dioxide [75–77]. The idea of using pure N2 as the sparging gas was initially taken into consideration. Using a mixture of N2 and CO2 for the conduction of the experiments was based on previous studies with R. albus in which pure CO2 was used in the initial atmosphere of the experiments [23,39,47] as well as studies from which it was shown that the use of gas mixtures of N2 and CO2 and pure CO2, can lead to increased hydrogen yields in comparison to those obtained when pure N2 was used [49]. Nevertheless, it is can be assumed that although the factor of PCO2 was not taken into account, in general the proposed model describes successfully the metabolic path illustrated in Fig. 1, and supports strongly the assumption that R. albus metabolizes glucose via the Enterobacteriaceae pathway.

international journal of hydrogen energy 34 (2009) 3697–3709

5.

Conclusions [11]

In the present study a quantitative kinetic model was developed in order to describe hydrogen production from the bacterium R. albus during its growth on glucose. Based on the reactions of glucose catabolism according to the Enterobacteriaceae type pathway, proper mathematical equations were developed taking into account the interactions among metabolites and the effect of hydrogen partial pressure on formate degradation. Subsequently the equations were incorporated into the process of acidogenesis of the Anaerobic Digestion Model 1 (ADM1) whereas the processes of hydrolysis, acetogenesis from volatile fatty acids and methanogenesis were excluded from the model. Using the computer program Aquasim as a modeling platform, the experimental data of batch experiments with different initial concentrations of glucose and different initial hydrogen partial pressures were simulated and the kinetic constants of the process were estimated. It was assumed that hydrogen partial pressure has a significant effect on the degradation of formate whereas it does not directly affect acetate and ethanol production. Subsequently, the accuracy of the model was verified by successfully simulating the performance of a CSTR in all hydraulic retention times tested.

[12] [13] [14] [15] [16] [17] [18] [19] [20]

[21]

[22]

Acknowledgements [23]

The authors wish to thank the Hellenic General Secretariat for Research and Technology for the financial support of this work under ‘‘PENED 2001, 01ED390’’.

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