Substrate consumption rates for hydrogen production by Rhodobacter sphaeroidesin a column photobioreactor

iiii

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 103-113

Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor [nci Eroglu a

a,, Kadir Asian, Ufuk Giindiiz b Meral Yticel b Lemi Tiirker c

Department of Chemical Engineer&g, Middle East Technical University, 06531 Ankara, Turkey b Department of Biology, Middle East Technical University, 06531 Ankara, Turkey c Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey

Received 13 October 1998; received in revised form 4 December 1998; accepted 22 December 1998

Abstract

The effect of L-malic acid and sodium glutamate, which serve as the carbon and nitrogen source, respectively, on hydrogen production by Rhodobacter sphaeroides O.U.001 has been investigated in a batch water jacketed glass column photobioreactor (PBR), which has an inner volume of 400 ml. The PBR was operated at different carbon to nitrogen ratios at 32~ with a tungsten lamp at a light intensity of 200 W m-2. Carbon to nitrogen ratio was found to be an important parameter for bio-hydrogen production. Moreover, hydrogen gas production was dependent on certain threshold concentrations of sodium glutamate. L-malic acid consumption was found to be first order with respect to L-malic acid concentration, whereas sodium glutamate consumption was found to be second order with respect to glutamate concentration. It was concluded that there is a close relationship between the hydrogen production rate and substrate consumption rates. A kinetic model is developed, which relates hydrogen gas production per amount of biomass, L-malic acid, and sodium glutamate concentrations. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen production; Photobioreactor; Rhodobacter sphaeroides; Instantaneous fractional yield; Hydrogen production factor; Dual substrate

1. Introduction

By using sunlight, some photosynthetic microorganisms produce hydrogen. Hydrogen is considered to be a promising, ideal, and renew-

* Corresponding author. Tel.: + 90-312-2102609; fax: + 90312-2101264. E-mail address: [email protected] (i. Eroglu)

able form of energy. Hydrogen is also used industrially in the synthesis of ammonia, in hydrogenation reactions, and in many other important applications. Bioproduction of hydrogen by photosynthetic bacteria from various substrates and from wastes has previously been investigated. Sasikala et al. (1991, 1992, 1995) produced hydrogen by Rhodobacter sphaeroides O.U.001. Recently, bioprocesses have been developed for hydrogen production by R. sphaeroides RV (Tsy-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: SO 168-1656(99)00064-4

104

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

gankov et al., 1993; Minami, 1997; Kitajima et al., 1998). At the Middle East Technical University in Turkey, three different bio-systems were designed for hydrogen production: (1) by R. sphaeroides O.U.001, (2) by coupled systems of Halobacterium halobium and E. toll, and (3) photoelectrochemical hydrogen production by H. halobium. In previous work (Arik et al., 1996), a 150 ml glass column photobioreactor (PBR) was constructed for the production of hydrogen by R. sphaeroides O.U.001 under anaerobic conditions and with a fixed light intensity. The optimum hydrogen production conditions were determined to be as follows: pH between 7.3 and 7.8, temperature 31-36~ light intensity 200 W m -2, and cell concentration 1.6-1.8 g 1-1 (dry weight). The maximum hydrogen production rate obtained was 0.047 1 1-1 h-1 gas produced per unit volume of culture with 99% purity. Then, a continuous PBR was scaled up to 400 ml (Eroglu et al., 1998). It was found that both the growth medium and the cell concentration have an influence on photosynthetic hydrogen production. The effect of parameters such as cell concentration and dual substrate concentrations (g-malic acid and sodium glutamate) on hydrogen production rate should be determined. If the relationship between the hydrogen production rate and the substrate concentrations can be expressed in terms of kinetic models, it might be possible to achieve prolonged continuous hydrogen production in a larger column PBR. To date, no reference exists in the literature about the modeling of large scale hydrogen production. To achieve such modeling, primarily kinetic models that relate the consumption of the carbon source, the nitrogen source and the cell growth and hydrogen production rates are required. Therefore, the objective of the present study is to find relationships between the consumption rates of the two substrates--u-malic acid as a carbon source and sodium glutamate as a nitrogen s o u r c e ~ o n cell growth and hydrogen gas production. This paper presents an initial quantitative approach to describe kinetics of hydrogen production by photoheterotrophic bacteria.

2. Materials and methods

R. sphaeroides O.U.001 (DSM 5648) was grown under anaerobic and sterile conditions in a minimal medium of Biebl and Pfenning (1981). The growth medium contained n-malic acid as the carbon source and sodium glutamate as the nitrogen source, and a vitamin solution (thiamin and niacin; 0.0005 g 1-1). Temperature was 32~ The growth medium was illuminated using a tungsten lamp at a light intensity of 200 W m -2. The initial pH of the growth medium was 7. Argon gas was used to create anaerobic conditions. The column PBR was made up of a glass cylinder that had an inner volume of 400 ml and was surrounded by a water jacket. At the top of the reactor, there was an inlet for the medium and outlets for the argon and for the hydrogen gas that was collected in a gas-measuring burette. Fresh medium was added from a reservoir that was placed above the PBR. Microorganisms were inoculated through the septum. At the bottom of the column PBR, there was an outlet for the culture and an inlet for argon gas. Experiments were carried out using media containing different initial amounts of the substrates n-malic acid and sodium glutamate. In the various experiments the initial concentrations of u-malic acid were 7.5, 15 or 30 mM, whereas the initial concentrations of sodium glutamate were 1, 2 or 10 mM. Samples were taken at 12 h time intervals while flushing the column with argon. Bacterial cell concentrations were measured as an increase in absorbance at 660 nm (Hitachi Spectrometer). The pH of the samples was also measured. In order to determine the consumption of the substrates, the samples were centrifuged and the supernatant was subjected to HPLC analysis (Shimadzu HPLC, BIORAD Aminex Ion Exclusion Column). The gas produced was analyzed by gas chromatography (Hewlett Packard 5890, Series II). We replicated each of the nine runs at least twice.

3. Results and discussion

In a set of nine replicated experiments, different combinations of initial substrate concentrations of

105

I. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

L-malic acid (CLMAo) and sodium glutamate (CNGo) were tested. The experiments showed that hydrogen production had started at some time after the inoculation of the bacteria. Different hydrogen production starting times (to) were obtained in different runs. Table 1 summarizes the total volume of hydrogen gas evolved (VT), gas production starting time (to), duration of the gas production, and the maximum cell concentration measured in these runs. Fig. 1 illustrates total hydrogen gas produced with respect to time in Runs 4 - 6 where initial L-malic acid concentration was 15 mM in each run. Initial sodium glutamate concentrations were varied and set at 1, 2 and l0 mM, respectively. High initial concentration of sodium glutamate (10 mM) in Run 6 led to a 4-fold decrease in hydrogen production relative to Run 4. Fig. 2 shows the growth curves of R. sphaeroides O.U.001 obtained for the same set of runs. The maximum cell concentration obtained in Run 6 was almost two times greater than that of Run 4 and Run 5. It was observed that excess sodium glutamate enhanced the cell growth but inhibited the hydrogen production. The pH varied between 7 and 7.6 in these runs. That is, during the course of these experiments, a slight decrease is observed in the pH of the medium during the

cell growth period, but once the hydrogen was produced, the pH increased. A similar comparison was made between the results obtained in Run 1, Run 4 and Run 7. In all these experiments the initial sodium glutamate concentration was set at 1 mM, and the initial L-malic acid concentrations were varied to 7.5, 15 and 30 mM, respectively. The total volume of hydrogen gas evolved, the duration of the hydrogen production, and the maximum cell concentration were increased, as the initial L-malic acid concentration was increased. This might indicate that high L-malic acid concentration enhanced both the cell growth and the hydrogen production at low sodium glutamate concentrations (Run 1 and Run 4). The pH varied between 7 and 7.8 during those runs. The consumption of the substrates during the runs must also be taken into account. It was observed that hydrogen production ceased when the L-malic acid had been totally consumed in all those runs having low initial sodium glutamate concentration (Runs 1, 2, 4, 5, 7 and 8) (Eroglu et al., 1998). However, if high sodium glutamate was present in the system (Runs 3, 6 and 9), both substrates were not utilized completely and they were left in the culture when hydrogen production stopped. It should be emphasized that cell concen-

g ,,,,,,,

200

0

--4~--.. 1 mM

100

'-"

2mM

. - ~ k ~ l O mM "

0

-----

----t

30

~--

;-

f

60

,,~ 90

f 120

~ 150

,I 180

t

210

240

Time (hrs)

Fig. 1. Total hydrogen production at a substrate concentration of 15 mM L-malic acid and three different sodium glutamate concentrations (1, 2 and l0 raM).

106

I. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

|

0

~

~6

0

~.o 0

~

~

0 ,..

~ 0

r

r

u ~

= E 0 f2~

~

~

L.

e~

t~

e.q

c.q

,,e1.

~

c'q

l~

,--

,..

r

~

,-.

c,q

~

,--

C,q ~

tg~

t~

tt~

|

0

0

0

f2~

0

|

e~

0 0

0

e~ ,_ .

0

F. ,

o ~,

"~,

~

"E .~_

107

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113 12 - - e - - 1 mM

.J

~10

~2mM

3=

--~ir-- 10 rr~

~'8 "t3

~6 u C

uo 4 u2 o ~==='~'~ 0

i

~

t

i

50

100

150

200

Time(hr)

250

Fig. 2. Growth curves of bacteria at a substrate concentration of 15 mM L-malic acid and three different sodium glutamate concentrations (1, 2 and 10 mM). tration reached very high values in a short time in these runs. That might indicate the positive effect of high sodium glutamate concentration on cell growth, but would also indicate the inverse effect on hydrogen production.

4. Dual substrate consumption rates Integral method of analysis followed, to interpret the substrate consumption data and to find the consumption rate equations for L-malic acid and sodium glutamate. During this analysis, the volume of the PBR was assumed to be constant, since small amounts of samples (2 ml) were taken out. Significant variations of temperature and concentration were not expected in the reactor, since the reactor was small and argon gas bubbled during sampling. The cells were suspended in the column, but some of the cells--most probably the dead cells--sank to the bottom, which caused small fluctuations in cell concentration values. Various rate models m including Monod type of equations--have been tried using the Microsoft Excel 5 package program. The following rate equations gave the best fit: the first order consumption rate equation for L-malic acid (Fig. 3):

(1)

-- rLMA = --dCLMA/dt = k1CLMA and the second order consumption sodium glutamate (Fig. 4):

rate

for

- ryG = -- dCyG/dt = k 2 C 2 G

(2)

Table 2 summarizes the best fitting rate parameters of Eqs. (1) and (2), where n denotes the order of the rate equation, kl and k2 are the rate constants for the first order (h-1) and for the second order (1 mol-1 h - l ) rate equations, respectively. R 2 measures the dispersion of distribution from the mean. It varies between values 0 and 1, where 1 denotes the maximum agreement of the experimental data to calculated data. It should be noted that these fits were quite acceptable, considering the experimental difficulties and the scatter in the experimental data. No direct relationship was found between the rate constants and the concentration of the other substrate. Therefore, these rate equations were considered to be independent. The second order consumption rate of sodium glutamate may indicate that two molecules of glutamate are involved in the consumption reaction as would be the case if the enzyme had two binding sites for glutamate.

5. Cell growth without hydrogen production The growth curves obtained were examined in two phases. Phase 1 is the cell growth without hydrogen production, and Phase 2 is the hydrogen production. In Phase 1, between initial time and tc, substrates were mainly consumed by the

108

I. Eroglu et al. / Journal of Biotechnology 70 (1999) 103-113

perimental data. For convenience in evaluating cell concentration and substrate distribution, two yield terms are introduced: instantaneous fractional yield of cells, y, and overall fractional yield of cells, Y. Let y,V/LMA(I) be the ratio of the rate of new cells formed to the rate of consumption of L-malic acid (in terms of mass) at any time t. YX/LMA is called instantaneous fractional yield of cells with respect to L-malic acid. Similarly, the instantaneous fractional yield of cells with respect to sodium glutamate, y x / N G ( t ) , is the ratio of the rate of mass of new cells formed to the rate of consumption of sodium glutamate (in terms of mass). The L-malic acid consumption rate, rLMA, and sodium glutamate consumption rate, -rNG, are related to the growth rate, rG, by yield factors YX/LMA and YX/NG. From Eqs. (1) and (3) and similarly from Eqs. (2) and (3), the following relations are obtained:

growing cells. The growth rate of the bacteria, rG (the change in cell concentration relative to the change in time) is: rG = d X / d t

= mm](

(3)

where X is cell concentration (g 1-1), t is time (h), and m m is the specific growth rate (h-1). Eq. (3) should be valid for the exponential growth phase of the bacteria. In this phase, the cells adapt themselves to their new environment. After the adaptation period, cells should multiply rapidly, and cell mass and cell number density should increase exponentially with time. This is a period of balanced growth in which all components of the cell grow at the same rate. That is, the average composition of a single cell remains approximately constant during this phase of growth. During balanced growth, the specific growth rate determined from either cell number or cell mass would be the same. Since the nutrient concentrations are large in this phase, the growth rate is assumed not to be limited by the nutrient concentration, hence the exponential growth rate could be expressed as first order. Time required for the inoculation of the bacteria is neglected. A plot of In X versus time yields mm which is the slope of the best fitting line to represent the cell growth data obtained for each run. It was interesting to note that mr, varied between 0.022 and 0.217 h for different runs (Table 2). This result might indicate the significance of dual initial substrate concentrations on specific growth rate. In the present work, a new approach, the yield model, has been introduced to represent the ex-

yX/LMA(I) = m m X / ( k , C L M A M W L M A )

(4)

yx/yG(t) = mmX/(keC2GMWNG)

(5)

The instantaneous fractional yields of cells with respect to each substrate depend on time. Therefore, they should be estimated by introducing the concentrations measured at that time. Table 3 lists the instantaneous yield factors estimated at to. Overall fractional yield which is the ratio of total mass of new cells formed until tc to total mass of L-malic acid consumed until tr is the mean of the instantaneous fractional yields between the initial time and t~ with respect to each substrate:

2,50

=oo gx I

y

l

-.- 2,00 1,50 1,00 '~' 0,50 0,00

~-

0

~

t

20

"

'

?"

40

I

I

I '

I

60 80 timelhour)

'

1

I

100

120

~

w

140

Fig. 3. The first order consumption rate model for L-malic acid in the medium containing 7.5 mM L-malic acid and 1 mM sodium glutamate initially.

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

109

2000 1600 ~'

1200

z

y = 4,7357x + 951 RZ = 0,8825

800 400

0

20

40

60 80 time (hour)

100

120

140

Fig. 4. The second order consumption rate model for sodium glutamate in the medium containing 7.5 mM L-malic acid and 1 mM sodium glutamate initially. X(tc) - Xo

YX/LMA = ( C N ~ o -

CN~(to)MWLMA)

(6)

Similarly, the overall fractional yield of cells with respect to sodium glutamate is: X(tc) - ;Co

YX/N~ = ( CNGo - CN~(tr

(7)

where Xo is the initial cell concentration, which is neglected in the present work. Table 3 lists the overall fractional yield factors estimated in Phase 1. The instantaneous fractional yields estimated at tr were greater than the overall fractional yields. The yields for L-malic acid were much less than the yields for sodium glutamate. Those results indicated the importance of sodium glutamate for cell growth.

6. Hydrogen production Hydrogen production rate is defined in three different ways: 1. The average hydrogen production rate per culture volume, Rn2av, which is calculated by dividing total volume of gas produced by the volume of the reactor and by the duration of gas production, and has the unit of l 1-1 h-z. 2. The maximum hydrogen production rate per culture volume, RH, which is estimated from the total volume of~gas produced versus time data by simulation with a polynomial curve fitting and differentiation with respect to time. The plot of hydrogen production rate versus time gives a maximum at some time, tm. This is the time corresponding to the maximum

hydrogen production rate per unit volume of the culture, where the units are 1 1-1 h-1. 3. The maximum hydrogen production rate per biomass, R h2, is calculated by dividing the maximum hydrogen production rate, RH2, by cell concentration, X, measured at /m, where the unit is 1 g-~ h-~. The maximum hydrogen production rates were found to be quite close to the average hydrogen production rates (Table 3), which might indicate that the hydrogen production rate does not change too much during the gas's evolution. The rate of hydrogen production obtained in Run 5 was the highest compared to the other runs (0.01 1 1-~ h - t or 0.0024 1 g - ~ h - ~). These results are quite comparable with the rates obtained by Kitajima et al. (1998) who found hydrogen production rates as 0.042 1 1-~ h-~ or 0.0042 1 g-~ h-~ in plane type photosynthetic bioreactor. Minami (1997) reported 0.015 1 1-~ h-~ for R. s p h a e r o i d e s RV in 10 1 continuous PBR, and 0.011 1 1-~ h for 1.4 1 continuous PBR. The cells grow at a slower rate after tc during hydrogen production. The cell growth rate in Phase 2 is: !

ro = d X / d t = m m X

(8)

where m m is the specific growth rate during hydrogen production, m m values were obtained from the slope of the best fitting line of In X versus time, between tc < t < tm, and are listed in Table 2. m m values were found to be almost an order of magnitude less than the mm values for Phase 1. That could be attributed to the increase in the rate of death of the cells. In continuous experi-

110

]. Eroglu et al. / Journal of Biotechnology 70 (1999) 103- 113

ments the difference between m m and mm is very critical. If the dilution rate is not adjusted to mm, the cells will wash out. Instantaneous fractional yields in Phase 2 were estimated from Eqs. (4) and (5) by replacing m m with mm, which might also involve the consumption of the substrates due to the maintenance of the cells and hydrogen production.

YHJx = 3.17(X/CNG2) T M

(R2=0.74)

(b) The interactive model: hydrogen production factor depends on the yield of both of the substrates. That is:

Y n J x oc (Yx/NG)/(Yx/LMA)

(13)

where Y n J x is proportional to (CLMA/CNG2). The best fitting equation is:

Y n J x = 3.48(CLMA/CNG2) ~

7. Yield models In order to compare the results of different runs, a factor (the hydrogen production factor) is defined:

yH2/X = R'H2tm

(12)

(R 2 = 0.82)

(14)

It is concluded that hydrogen production is related to the ratio of the concentration of Lmalic acid to the square of the concentration of sodium glutamate.

(9)

yn2/x, by definition, has the unit volume of gas produced per dry weight of bacteria (1 g-~). Table 3 lists the instantaneous fractional yields and the hydrogen production factor estimated at t m . Two yield models are proposed for the estimation of the hydrogen production factor: (a) The non-interactive model: the hydrogen production factor depends on the yield of one of the substrates: either on the yield of L-malic acid or on the yield of sodium glutamate: yH2/X W.YX/LMA OCX~ CLMA

(1 O)

YH2/X OCYNG/X Ct?.X/ CNG2

(1 1)

The hydrogen production factor did not depend ( X / C L M A ) , hence, R 2 was very small. However, the hydrogen production factor depended on (X/ CNG:). The best fitting equation is: on

8. Hydrogen production models considering carbon to nitrogen ratio It was observed that the hydrogen production rate was affected by the ratio of substrate consumption rates, as explained in the previous section. Moreover, the ratio of carbon to nitrogen could be taken as a parameter instead of the substrate concentrations individually (Minami, 1997). That is to say, the hydrogen production rate is related with residence time, the carbon to nitrogen ratio, and cell concentration. Using these parameters in trial runs, it was found that the following models give the largest R 2 values:

yH2/X = 6.25[(C/N)X] ~

(R 2 = 0.56)

Y n J x = 22.6[(C4/N)X] ~

(15)

(R 2 = 0.75)

(16)

Table 2 Summary of the rate parameters Run

k 1 (h -l)

R2

k2 (1 mol -I h -l)

R2

mm (h-1)

mm (h-1)

1 2 3 4 5 6 7 8 9

0.0179 0.0316 0.0089 0.0109 0.0183 0.0141 0.0075 0.0096 0.0101

0.92 0.94 0.96 0.90 0.88 0.91 0.87 0.87 0.70

4.74 3.43 0.19 2.68 2.45 0.27 2.78 8.12 0.30

0.88 0.63 0.86 0.72 0.95 0.89 0.79 0.82 0.73

0.058 0.038 0.217 0.068 0.164 0.089 0.072 0.022 0.14

0.0066 0.0061 0.0085 0.0021 0.0014 0.0286 0.0034 0.0020 0.0055

I. Eroglu et al. /Journal o f Biotechnology 70 (1999) 1 0 3 - 1 1 3

Z ee~

ec~ ~'.q tt~

o

o

,~i.

~,0

r162

o

o

o

fi

,d

I

fi

D" e~o

i

o

I

fi

E Z ~D < 0 .,,.,

,d

"0 0 ",a

~7 0

Z

r~ 0

..3 r

az~

o

o

c5

111

112

]. Eroglu et al./Journal of Biotechnology 70 (1999) I03-113

where C/N denotes the ratio of the total moles of carbon per the total moles of nitrogen present in the culture, whereas C4/N denotes the moles of carbon present in e-malic acid per the total moles of nitrogen present in the culture. It is interesting to note that hydrogen production depends on the moles of carbon present in L-malic acid more than on the total moles of carbon present in the medium.

9. Conclusions The following conclusions can be drawn from this study: 1. Both of the substrates (e-malic acid and sodium glutamate) are vital for hydrogen production. Moreover, hydrogen gas production is dependent on certain threshold concentrations of sodium glutamate. Interestingly, as the concentration of sodium glutamate reaches a certain upper limit, hydrogen production ceases. Therefore, the stress condition exerted by sodium glutamate to produce hydrogen is operative in a certain range of the concentration. 2. Hydrogen production starts after a lag period (40-80 h) and continues even after, the cell concentration has leveled out. The specific growth rate of bacteria in the hydrogen production phase is less than the specific growth rate of the bacteria in the exponential cell growth phase without hydrogen production. This finding contradicts the conclusions of previous researchers (Sasikala et al., 1992; Arik et al., 1996), who found that hydrogen was mainly produced in the exponential phase of growth in smaller sized PBRs. 3. The e-malic acid consumption rate was found to be first order with respect to the e-malic acid concentration, whereas the sodium glutamate consumption rate was found to be second order with respect to the sodium glutamate concentration. 4. There is a relationship between the cell concentration and the hydrogen production rate. However, the hydrogen production rate basically depends on the L-malic acid to sodium

glutamate ratio. The maximum hydrogen production rate is observed with the growth medium initially containing 15 mM L-malic acid and 2 mM sodium glutamate concentrations.

Acknowledgements This research has been supported by the Turkish Scientific Research Council (TUBITAK) Project number TBAG 1535, and the Middle East Technical University (METU) Research Fund, project number AFP-96-07-02-02.

Appendix A. Nomenclature

C C/N C4/N kl k2 mm t

mm

MW RG R RH 2

Rh2 RH2av R2 t tc tm

concentration (mol 1-1) ratio of the total moles of carbon per moles of nitrogen present in the culture ratio of the moles of carbon present in L-malic acid per moles of nitrogen in the culture first order reaction rate constant (h -1) second order reaction rate constant (1 mol-1 h-l) specific growth rate in Phase 1 (h-') specific growth rate in Phase 2 (h -1) Molecular weight (g mol -~) growth rate of the bacteria (g 1h -l) consumption rate (mol 1-~ h-l) maximum hydrogen production rate per culture (1 1-~ h -~) maximum hydrogen production rate per biomass (1 g-1 h-l) average hydrogen production rate per culture (1 1-~ h -~) goodness of fit time (h) hydrogen gas production starting time (h) residence time of maximum hydrogen production rate (h)

i. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

Vv X Xmax

total volume of hydrogen gas evolved (ml) cell dry weight concentration (g 1-1 ) maximum cell concentration (g 1-1)

yH2/X

hydrogen production factor (1 g-l)

y

instantaneous fractional yield of cells overall fractional yield of cells

Y

Subscripts H2 LMA NG o

X

hydrogen gas L-malic acid sodium glutamate initial cells

References

Arik, T., Gunduz, U., Yucel, M., Turker, L., Sediroglu, V., Eroglu, I., 1996. Photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001, Proceedings of the 11th World Hydrogen Energy Conference, Stuttgart, Germany, vol. 3, 2417-2424.

113

Biebl, H., Pfenning, N., 1981. Isolation of member of the family Rhodosprillacaea. In: The Prokaryotes. SpringerVerlag, New York, pp. 267-273. Eroglu, I., Asian, K., Gfindfiz, U., Yficel, M., Tfirker, L., 1998. Continuos hydrogen production by Rhodobacter sphaeroides O.U.001. In: Zaborsky, O.R. (Ed.), BioHydrogen. Plenum press, New York, pp. 143-149. Kitajima, Y., E1-Shishtalwy, R.M.A., Ueno, Y., Otsuka, S., Miyake, J., Morimoto, M., 1998. Analysis of compensation points of light using plain type photosynthetic bioreactor. In: Zaborsky, O.R. (Ed.), BioHydrogen. Plenum Press, New York, pp. 359-367. Minami, M., 1997. Biohydrogen production using sewage sludge by photosynthetic bacteria. Paper presented in BioHydrogen '97, the International Conference on Biological Hydrogen Poduction, Kona, Hawaii, USA. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1991. Environmental regulation for optimal biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 16, 597-601. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1992. Photoproduction of hydrogen from the waste water of a distillery by Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 17, 23-27. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1995. Regulation of simultaneous hydrogen photoproduction during growth by pH and glutamate in Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 20, 123-126. Tsygankov, A.A., Hirata, Y., Miyake, M., Asada, Y., Miyake, J., 1993. Hydrogen evolution photosynthetic bacterium Rhodobacter sphaeroides RV immobilized on porous glass. New Energy Systems and Conversions, Universal Academy Press, pp. 229- 233.