Biotechnol Lett (2006) 28:831835 DOI 10.

1007/s10529-006-9008-8

ORIGINAL PAPER

Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae

Biswajit Mandal Kaushik Nath Debabrata Das

Received: 20 January 2006 / Accepted: 17 February 2006 / Published online: 2 June 2006 Springer Science+Business Media B.V. 2006

Abstract When the partial pressure of H2 was decreased by lowering the total pressure in the headspace of the reactor in a batch fermentation process from 760 mm Hg to 380 mm Hg containing Enterobacter cloacae, the molar yield of H2 increased from 1.9 mol to 3.9 mol H2/mol glucose. The maximum production rate was 0.017 mmol H2/h l at 380 mm Hg. The lag period as well as total batch time of H2 production decreased using a decreased partial pressure. Keywords Biohydrogen Enterobacter cloacae fermentation H2 yield partial pressure

yield of H2. This is one of the major deterrents of the fermentative H2 production process. At most a maximum of 4 mol H2 could be obtained per mol glucose during acetate fermentation. To address this problem, efforts are needed to improve the operating conditions to overcome thermodynamic limitations (Fabiano et al. 2002) of the acetate fermentation reaction represented below. C6 H12 O6 2H2 O ! 2CH3 COOH2CO2 " 4H2 " A 2B 2C 2D 4E

Introduction Major advantages of fermentative H2 production processes are higher evolution rate of H2 and a wide gamut of substrate utilization. However, the inherent disadvantage of these processes is lower

Decrease of H2 partial pressure could be considered as an approach towards improvement of H2 productivity (Levin et al. 2004). Equilibrium constant of the above reaction is Kc C2 D2 E4 AB2 1

B. Mandal K. Nath D. Das (&) FermentationTechnology Laboratory, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India e-mail: ddas@hijli.iitkgp.ernet.in K. Nath Department of Chemical Engineering, G H Patel College of Engg & Technology, Vallabh Vidyanagar 388120 Gujarat, India

According to Le Chateliers principle, the equilibrium of the above reaction will shift to the right if one or both of the gaseous products of the reaction (D or E) is removed. Decrease in partial pressure of H2 (E) by reducing total pressure of the system allows the reaction equilibrium to shift towards right side and thereby enhance the H2 production. In general, biological H2 production from organic substrates is limited by the thermodynamics

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of the hydrogenase reaction, involving the enzyme-catalyzed transfer of electrons from an intracellular electron carrier molecule to protons. On the contrary, protons are poor electron acceptors ( EH2 414 mV); so, the electron donor must be a strong electron reducing agent. Ferredoxin is a low-potential (EFd = )400 mV) ironsulfur containing protein that is capable of reducing proton to H2. Another important intracellular electron carrier, NADH, has a higher redox potential (ENADH = )320 mV). Under actual conditions the ability of reduced ferredoxin and NADH to reduce protons is determined by the redox potential of the overall reaction. Assuming the intracellular concentrations of the oxidized and reduced form of ferredoxin and NADH are equal, H2 production becomes thermodynamically unfavorable at high H2 partial pressure, this can be determined by the following correlation (Largus et al. 2004): PH2 ;max exp f2FEH2 Ex =RTg 2

lowering the operating pressure substantially improves H2 production in an anaerobic, dark fermentation process. The molar yield of H2, its production rate, and lag phase of the gas production under reduced operating pressure are reported.

Materials and methods Microorganisms and growth Enterobacter cloacae DM 11, a H2-producing bacterium, was grown overnight at 37 1C with shaking at 200 rpm in nutrient broth at pH 6.0 (Kumar et al. 2001). The partial pressure of H2 gas was reduced by decreasing the operating pressure of the reactor by adjusting the saline level of the gas collector using a peristaltic pump, which was measured by a U-tube mercury manometer connected with the system (Fig. 1). This was inoculated with 20% (v/v) of overnight culture of E. cloacae DM 11 into MYG medium containing 1% (w/v) malt extract, 0.4% (w/v) yeast extract and 1% (w/v) glucose. After inoculation, the reactor (see Fig. 1) was ushed with Ar gas (99.99%) for 5 min to ensure anoxic conditions. The medium was stirred at 100 rpm. The evolved gas from the headspace of the reactor was passed though 30% (w/v) of KOH to remove CO2 from the gas stream. Inoculation was continued until gas production ceased. H2 production was monitored in a gas collector by water displacement method at room temperature and at atmospheric pressure. The experimental data points are the average of minimum duplicate results, with variation not exceeding 5%. Analytical methods The composition of the gas was analyzed by thermal conductivity detector (GC) using a 80/ 100 Porapak-Q (3.2 mm diam. 2 m) column. The oven, injector and detector were held at 80C, 150C and 200C respectively. N2 was used as the carrier gas at 20 ml/min. For each batch the gas samples were analyzed twice. Biomass was measured turbidometrically at 600 nm. Reducing

where Ex is the redox potential of electron donor, F is the Faradays constant, R is the ideal gas constant, and T is the absolute temperature. This correlation indicates that for ferredoxin, H2 production can continue as long as the partial pressure of H2 is less than @0.3 atm (3 104 Pa): for NADH, the partial pressure of H2 must be less than @6 10)4 atm (60 Pa). This implies that at a very low partial pressure ( < 60 Pa), NADH could also be used for H2 production. The rst attempt to reduce the partial pressure was to use KOH to remove CO2 in the headspace, which decreased the partial pressure of H2 by 87% (Wooshin et al. 2005). This enhanced the yield of H2 from 1.4 mol to 2 mol H2/mol glucose. Gas sparging is another useful technique to decrease the H2 partial pressure to enhance its yield. Mizuno et al. (2000) observed that the specic H2 production rate increased from 1.5 ml H2/min g biomass to 3.1 ml H2/min g biomass under N2 sparging conditions. Yields of H2 using various microorganisms and process conditions are presented in Table 1 which shows that the yield of H2 in most of the processes is below the maximum theoretical value. The present investigation shows, for the rst time, that reduction of H2 partial pressure by

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Biotechnol Lett (2006) 28:831835 Table 1 Comparative studies on the H2 yield using different microbial strains and different processes Organisms Rhodopseudomonas palustris P4 Enterobacter aerogens Enterobacter cloacae /Emphasis> IIT BT 08 Citrobacter sp. Y19 Clostrium thermolacticum Enterobacter cloacae DM11 Substrates Glucose Molasses Glucose Glucose Lactose Glucose Process Batch, with intermittent purging of Ar Ar sparging, batch Continuous (Immobilized bioreactor) Batch Ar sparging Batch (using KOH as scavenger) Batch at operating pressure 380 mm of Hg (initial sparging with Ar) Maximum yield (mol H2/mol substrate) 2.8 1.6 2.3 2.5 2.13.0 3.9 References Oh et al. (2002)

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Tanisho et al. (1998) Kumar and Das (2000) Oh et al. (2003) Collect et al. (2004) Present study

Fig. 1 Schematic diagram of the experimental set-up 1 Shake ask (300 ml medium); 2 Hg manometer; 3 liquid trap; 4 CO2 absorber [50 ml 30% (w/v) KOH]; 5 Gas collector (2000 ml); 6 Peristaltic pump; 7 Pressure controller; 8 & 9 Saline water storage

sugar (glucose) content of the medium was estimated spectrophotometrically using DNS dinitosalicylic acid method.

Results and discussion Studies were conducted to observe the effect of reduced partial pressure of H2 on the yield as well as production rate of H2. A perusal of Table 2 reveals that reduction of operating pressure of the reactor vessel could substantially increase the production of H2. The molar yield of H2 enhanced from 1.9 mol to 3.9 mol H2/mol glucose, on lowering the operating pressure from 760 mm Hg to 380 mm Hg. Maximum production rate was recorded as 0.017 mmol/h l at an operating pressure of 380 mm Hg. The sharp increase of yield

has certain implications. Firstly, the reduction of total pressure in the reactor might have facilitated the equilibrium of the desired reaction thereby minimizing the likelihood of any sort of inhibition by unwanted solventogenic pathways. Secondly, the lower partial pressure of H2 by lowering operating pressure inhibits the consumption of H2 leading to the production of reduced by-products such as ethanol and/or organic acids (Oh et al. 2002). Since alcohol production involves the consumption of H2 in the form of reducing equivalents such as NADH, it is inevitable that fermentation conditions that favor the metabolism of sugar to alcohols reduce H2 production. Further, the stripping of gas favors increased dissolution driving force by increasing the pressure difference between the liquid phase and the gas phase in the headspace of the reactor. This results in increased rate of production of H2 in the system. Experimental results indicated that although the consumption of substrate gradually decreased with reduction of operating pressure (Table 2), it was not proportional with H2 production. These imply that regulation of metabolic pathway is rather more important for increased H2 production. Consistent increase in H2 yield from glucose by lowering operating pressure focuses the requirement to optimize the same for maximum H2 yield. Moreover, as CO2 is absorbed in KOH solution, the overall ratio of CO2 to H2 content of the gas decreases. It merits mentioning that the reduction of concentration of both the gas components was particularly important, because homoacetogenesis reaction is generally

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834 Table 2 Process parameters of H2 production under different operating pressurea Operating pressure (mm Hg) 760 710 660 550 380 330
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Biotechnol Lett (2006) 28:831835

Amount of glucose consumed (g) 2.0 1.9 1.9 1.9 1.7 2.0

Maximum volumetric production at STP (ml) 552 891 986 1032 1074 830

Maximum production rate (lmol/h l) 6 10 15 15 17 12

Maximum yield obtained (mol H2/mol substrate) 1.9 3.2 3.6 3.7 3.9 2.9

Substrate: Glucose (initial amount of glucose is 3 g)

Process: Batch, Initially ushed with Ar (99.99%) Microorganism: Enterobacter cloacae DM11 Working volume: 300 ml STP: Standard temperature and pressure

associated with concentration of H2 and CO2 present in gas produced. Reports state that, lowering the concentration of both these gaseous components inhibits this homoacetogenesis reaction, as represented by (Oh et al. 2003). 4H2 2CO2 ! CH3 COOH 2H2 O Additional signicant feature of reduction of operating pressure in the reactor was the decrease of initiation time for gas production (Table 3). This can be attributed to the decrease of gas solubility in the production medium at low pressure. At low pressure, the dissolved gases including dissolved oxygen, initially present in the production media might tend to escape to the headspace of the reaction vessel and be replaced by argon. Once production of gas starts, it escapes from liquid phase to the gas phase. Under these

conditions, the production media could be considered to remain as a homogeneous liquid system rather than heterogeneous gas-liquid system. This homogeneity of the medium might have facilitated the substrate utilization by microorganisms effectively. The decrease in batch time due to lowering of partial pressure of H2 might be due to the fact that the dissolved gases during production have not hindered the utilization of substrate, as the lowering of operating pressure enhanced the stripping of gas. Experiments were conducted to study the change of pH under different pressure. The results reveal that change of pH is less at low operating pressure. The might be due to fast utilization of substrate by cells but a less plausible explanation might be due to inhibition of the homoacetogenesis reaction responsible for acetic acid formation.

Table 3 Parameters listed after the complete dark fermentation under different operating pressurea Operating pressure (mm Hg) 760 710 660 550 380 330
a

Overall lag period (min) 90 60 30 20 15 15

Overall batch period (h) 17 15 12 12 11 13

Change in initial pH 1.5 1.5 1.4 1.3 1.3 1.4

Final cell mass concentration (g/l) 1.48 1.74 1.91 2.05 2.28 1.73

Substrate: Glucose (Initial amount of glucose is 2.98 g)

Process: Batch, Initially ushed with Ar (99.99%) Microorganism: Enterobacter cloacae DM11 Working volume: 300 ml

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Table 3 reveals that the nal cell mass concentration increased with decrease in the operating pressure. The results imply that reducing operating pressure of H2 facilitates the growth of cell. However, at 330 mm Hg operating pressure the nal cell mass concentration along with production rate and yield of H2 abruptly reduced. It is somewhat difcult to adduce any exact reasoning from the ndings presented in Table 3 as to why there is increase of cell mass concentration up to certain pressure and thereafter it decreases. One plausible reason of this can be attributed to the fact that at low operating pressure the risk of contamination increases resulting in undesired fermentation (Kimmons et al. 1999).

References
Collect C, Adler N, Schwitzguebel JP, Peringer P (2004) Hydrogen production by clostridium thermolacticum during continuous fermentation of lactose. Int J Hydrogen Energy 29:14791485 Fabiano B, Perego P (2002) Thermodynamic study and optimization of hydrogen production by Enterobacter aerogenes. Int J Hydrogen Energy 27:149156 Kimmons JE, Brown KH, Lartey A, Collison E, Mensah PP, Deway KG (1999) The effect of fermentation and/ or vacuum ask storage on the presence of coliforms foods prepared for Ghanaian children. Int J Food Sci Nutr 50:195201 Kumar N, Das D (2000) Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08. Process Biochem 35:589593 Kumar N, Ghosh A, Das D (2001) Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae. Biotechnol Letts 23:537541 Largus TA, Khursheed K, Muthanna H Al-Dahhan, Brian AW, Rosa DE (2004) Production of Bioenergy and biochemicals from industrial and agricultural wastewater. Trends Biotechnol 22:477485 Levin DB, Pitt L, Love M (2004) Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 29:173185 Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T (2000) Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol 73:5965 Oh YK, Seol EH, Yeol Lee E, Park S (2002) Fermentative hydrogen production by a new chemolithotrophic bacterium Rhodopseudomonas palustris P4. Int J Hydrogen Energy 27:13731379 Oh YK, Seol EH, Kim JR, Park S (2003) Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19. Int J Hydrogen Energy 28:13531359 Tanisho S, Kuromoto M, Kadokura N (1998) Effect of CO2 removal on hydrogen production by fermentation. Int J Hydrogen Energy 23:559563 Wooshin P, Seung HH, Sang-Eun OH, Logan BE, Kim IS (2005) Removal of Headspace CO2 Increases Biological Hydrogen Production. Environ Sci Technol 39:44164420

Conclusion Among the various approaches to decrease H2 partial pressure in a reactor, the lowering of operating pressure appears promising. An alternative approach to decrease the H2 partial pressure in a reactor is to sparge the reactor with argon. However, even occasional sparging dilutes the product, H2, and also increases the overall costs. In contrast, lowering of operating pressure in the reactor can maintain the purity of H2 gas. Higher production rates, high yields and a lower initiation time of H2 production are thereby achieved by the expenditure of minimum energy. Further research is now necessary to develop this laboratory study into a practical reality.
Acknowledgement Financial assistance received from the Department of Biotechnology, Ministry of Non-Conventional Energy Source, Ministry of Science and Technology, Government of India is thankfully acknowledged.

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