Biological hydrogen photoproduction using dairy and sugarcane waste waters

Bioresource Technology 48 (1994) 9-12 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94/$7.00 ELSEVIER

BIOLOGICAL H Y D R O G E N PHOTOPRODUCTION USING DAIRY A N D S U G A R C A N E WASTE WATERS A. Thangaraj & G. Kulandaivelu School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India (Received 18 May 1993; revised version received 2 September 1993; accepted 9 September 1993)

obtained from the Deutsche Sammlung Von Mikroorganismen, Germany and was grown in the liquid culture medium prescribed by Weaver et aL (1975), in tightly closed reagent bottles. The temperature was maintained at 30°C. The culture bottles were shaken twice a day to disperse the sedimented cells. The cultures were illuminated by a bank of cool white fluorescent lamps. Irradiance at the culture surface was 10 Wm-2. The unicellular cyanobacterium, Anacystis nidulans (IU 625), obtained from the University of Indiana Culture Collection, USA, was grown photoautotrophically in modified Allen and Arnon's liquid culture medium (Allen & Arnon, 1955) at 30°C. The culture was developed in sterile growth medium in conical flasks and agitated continuously using an orbital shaker. Illumination was provided by a bank of cool white fluorescent lamps at 10 W m-2 at the culture surface.

Abstract

Photoproduction of H 2 by the photosynthetic bacterium, Rhodopseudomonas, and the cyanobacterium Anacystis, grown in the dairy and sugarcane waste waters was investigated. The growth and H 2 production capacity of these organisms were maximum in 50-60% concentrations of the industrial effluents. Both Rhodopseudomonas and Anacystis cells showed 70--90% growth in the effluents and they couM produce as much as 50-60% 112 when compared to those grown in the normal culture media. Key words: Hydrogen photoproduction, photosynthetic bacteria, cyanobacteria, Rhodopseudomonas, Anacystis, industrial effluents. INTRODUCTION

Biological H 2 production has been gaining serious attention due to global energy crisis. Photosynthetic bacteria and cyanobacteria are notable for their capability of fixing nitrogen and evolving H 2. The photosynthetic non-sulphur bacteria have the capacity to use a wide range of biodegradable organic substances including carbohydrates, organic acids and fatty acids for their growth and H 2 production (Kumazawa & Mitsui, 1982). Industrial effluents are rich in such organic substances and essential inorganic materials. Hence, H 2 production system using photosynthetic bacteria and cyanobacteria with concomitant depletion of organic wastes and inorganic materials could serve a dual purpose (Sasikala et al., 1991 ). In the present study, the photosynthetic bacterium, Rhodopseudomonas, and the cyanobacterium, Anacystis, were grown in dairy and sugarcane effluents after suitable dilution. The growth and the H 2 production ability of the ceils grown in these effluents were monitored and compared with those grown in normal culture media.

Growth of the cells in waste waters

The effluents from dairy plant and sugarcane industry were collected and filtered first with Whatman No. 1 filter paper to remove the debris and then through 0.45 /~m filter (Millipore). The pH of the effluents was set to 6.8 and 7.6, respectively for Rhodopseudomonas and Anacystis. The chemical oxygen demand (COD) and the biological oxygen demand (BOD) were analysed

Table 1. Physico-chemical characters of dairy and sugarcane effluents"

Components Colour pH COD (mg/litre - 1) BOD (mg/fitre- 1) Na (mg/litre- 1) K (mg/litre- 1) Fe (mg/litre- 1) Cu (mg/litre- 1) Mn (mg/litre- l)

METHODS Organisms and growth conditions The purple non-sulphur photosynthetic bacterium,

Rhodopseudomonas

capsulata

(DSM

1710),

Dairy effluent

Sugarcane effluent

Cloudy white

Pale straw yellow 8"14 276 54 4.50 1.64 10"83

6.92 134 42 1.54 1.87 2.46 0.28

0.72

0.18

0.60

aFor details on various analyses see Materials and Methods.

was 9

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A. Thangaraj, G. Kulandaivelu

according to Standard Methods (APHA, 1985). The contents of Na, K, Fe, Cu and Mn were estimated by atomic absorption spectrophotometry (Table 1). Initially, to check the optimum concentration for the better growth of the organisms, the waste waters were diluted with sterile water. The optimum concentration of the waste waters for maximum growth was determined for further experiments. Cells having equal chlorophyll concentration were inoculated and maintained under normal culture conditions. Control cells were grown in normal culture media as described above.

Measurement of hydrogen production Rate of H2 evolution was measured by gas chromatography. Five millilitres of cell suspension were taken in a 12 ml vial and sealed with suba-seal. The gas phase was replaced with argon by repeated flushing of the cell suspension with argon. The vials were incubated in dark for 3 h and then transferred to light. Optimum light intensity and temperature were maintained. A known volume of gas was removed from the gas phase of the reaction vial with a gas-tight syringe and was injected into the gas chromatograph (Hewlett Packard, Model 5890, USA) fitted with carbosieve S-II column (310 x 0"3 cm, 100-120 mesh) and a thermal conductivity detector (TCD). Nitrogen was used as the carrier gas at a flow rate of 30 ml min-~. The oven, injection port and detector temperatures were maintained at 70, 100 and 150°C, respectively. The signal was recorded in a Hewlett Packard integrator (Model 3390A). The volume of H z was calculated using pure Hz as standard.

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-~

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v ol

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Sugarcane

Pigment analysis From the cells of Rhodopseudomonas, bacteriochlorophyll (BChl) was extracted with acetone:methanol (7:2, v/v). The absorbance of the extract was measured at 775 nm and the BChl content was calculated by the method of Cohen-Bazire et al. (1957). In Anacystis, pigment extract was made with 90% methanol. The absorbance of the extract was measured at 663 um and the concentration of chlorophyll a (Chl a) was estimated according to Mackinney ( 1941 ). RESULTS AND DISCUSSION

Optimum concentration of effluents for maximum growth and H 2 production The growth of the cells in waste waters at different dilutions is given in Fig. 1. Both Rhodopseudomonas and Anacystis cells grown in 50-60% concentrations of the industrial effluents exhibited maximum growth. Similar to the growth of cells, the H2 production ability of these organisms reached the maximum level when they were grown in 50-60% concentrations of the

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Fig. 1. Effect of dilution of waste water and growth of Anacystis (o o) and Rhodopseudomonas (o o) cells. Changes in the cell number was monitored in six dayold cultures. Values represent average of three individual measurements. I

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Growth measurement The number of cells in the suspension was determined by direct microscopic counting using a Neubauer improved double haemocytometer.

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Fig. 2. Effect of dilution of waste water on H 2 photoroduction by Anacystis (0 o) and Rhodopseudomonas e) cells. The amount of H 2 produced was determined using cells collected on the 6th day. Values represent the mean of three individual measurements. Scales on the left and right side indicate the rate of H 2 production by Anacystis and Rhodopseudomonas, respectively. waste waters (Fig. 2). Concentrations above and below this level notably reduced the growth and H 2 production efficiency of both the organisms. Since the waste waters collected from various sources were rich in organic substances, suitable dilution was necessary for the better growth and H z production of the organisms. Similar changes were also reported by Sasikala et al. (1991). The poor growth and low H 2 production at higher and lower concentrations might be due to the increased BOD and insufficient nutrients, respectively.

1-12photoproducfion using dairy and sugarcane waste waters Growth and H z production of ageing cultures Culture age is one of the major factors which influence the growth as well as the metabolic activities of an organism. To study the influence of culture age on growth and H e production, the growth pattern and H2 production efficiency of Rhodopseudomonas and Anacystis grown in waste waters were followed up to 30 days and the results were compared with those from cells grown in the normal culture media. Throughout the estimated period of 30 days, all the effluents used here supported the growth of the organisms. Both organisms showed gradual increase in cell numbers with increasing age (Figs 3(a) and (b)). Interestingly, in dairy effluent the growth rate followed those of the cultures grown in the culture medium and accounted for about 95%. Such high rate growth might be due to the rich nutrient content in these effluents (Sasikala et al., 1991) and also due to reduced BOD in the medium (Kumazawa & Mitsui, 1982). Unlike the rate of culture growth, the H e production ability of both the organisms was not linear in ageing cultures. Rhodopseudomonas cells grown in waste waters and in culture medium exhibited maximum H 2 production on

the eighth day. Thereafter, a gradual decrease in the rate was noted (Fig. 4(a)). Similarly, Anacystis showed the maximum H 2 production on the sixth day followed by a gradual decrease (Fig. 4(b)). Among the waste waters used, dairy effluent was found to be more suitable than others for growing of both Anacystis and Rhodopseudomonas for efficient H 2 photoproduction. It is well known that the rate of photosynthesis, respiration, N e fixation and other metabolic activities of photosynthetic bacteria and cyanobacteria have been shown to be influenced by the culture age, nutrient depletion, increased cell density and self-shading, and these factors in turn may affect the H e production ability. The decrease in the H e production ability in the later stages of growth might be due to the reduced light conversion ability (Phlips & Mitsui, 1983) and also due to the increased H2 uptake activity of the ageing cells (Ravi, 1987). Even though the growth and H 2 production ability of the waste water grown cells were less, 50-70% as compared to the culture media grown cells, this is highly significant from the economic point of

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(b) Changes in the growth of Anacystis (a) and Rhodopseudomonas (b) as a function of age of the culture. Cultures were developed either in nutrient medium (o o) and in 500 concentration of dairy (n n) and sugarcane (e --o) effluents. Values represent the mean of three measurements and are significant at + 5% level. Fig. 3.

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(b) Fig. 4. Relative n 2 photoproduction efficiency of Anacyst/s (a) and Rhodopseudomonas (b) cells at different stages of growth in the culture medium (o o) and in 50% concentration of dairy (m n) and sugarcane (o o) effluents. H 2 photoproduction was monitored gas chromatographically up to 30 days at regular intervals. Values represent the average of three individual measurements and are significant at + 5% level.

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A. Thangaraj, G. Kulandaivelu

view. A suitable modification of this technique would provide an economically viable means of biological H 2 photoproduction. REFERENCES

Allen, M. B. & Arnon, D. I. (1955). Studies on nitrogen fixing blue green algae. I: Growth and nitrogen fixation by Anabaena cylindrica Lemn. Plant Physiol., 30, 366-72. APHA (1985). Standard Methods for the Examination of Water and Waste Water (16th edn). American Public Health Association/American Water Works Association/ Water Pollution Control Federation, Washington, DC. Cohen-Bazire, G., Sistrom, W. R. & Stainer, R. Y. (1957). Kinetic studies of pigment synthesis by non-sulfur bacteria. J. Cell. Comp. Physiol., 49, 25-68. Kumazawa, S. & Mitsui, A. (1982). Hydrogen metabolism of photosynthetic bacteria and algae. In CRC Handbook of

Biosolar Resources, Vol. I, Basic Principles, Part 1, ed. A. Mitsui & C. C. Black. CRC Press Inc., Boca Raton, FL, pp. 209-22. Mackinney, G. (1941). Absorption of light by chlorophyll solutions. J. Biol. Chem., 140, 315-22. Phlips, E. J. & Mitsui, A. (1983). Role of light intensity and temperature in the regulation of hydrogen photoproduction by the marine cyanobacterium Oscillatoria sp. Miami BG7. Appl. Environ. Microbiol., 45, 1212-20. Ravi, V. (1987). Hydrogen photoproduction in higher plants, algae and bacteria: Identification of rate limiting steps. PhD thesis, Madurai Kamaraj University, India. Sasikala, K., Ramana, C. V. & Subramanyam, M. (1991). Photoproduction of hydrogen from waste water of a lactic acid fermentation plant by a purple non-sulfur photosynthetic bacterium, Rhodobactor sphaeroides O.U.001. Ind. J. Exp. Biol., 29, 74-5. Weaver, P. E, Wall, J. D. & Gest, H. (1975). Characterization of Rhodopseudomonas capsulata. Arch. Microbiol., 105, 207-16.