Effect of some physiological factors on nitrogenase activity and

Vol. 45, No. 2, June 1998

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages 245-253

EFFECT OF SOME PHYSIOLOGICAL FACTORS ON NITROGENASE ACTIVITY AND NITROGENASE MEDIATED HYDROGEN EVOLUTION BY MIXED MICROBIAL CULTURE

Ashok Kumar*, Seema R. Jain, Vipin C. Kalia and Arvind P. Joshi Centre f o r Biochemical Technology (CSIR), University Campus, Mall Road, Delhi-l l O007, India Received January 12, 1998

Summary. Fermentative H2 evolution, nitrogenase activity (acetylene reduction) and nitrogenase mediated H2 evolution was studied in free cells of mixed microbial population of H2 producers. At 3% glucose level, the cells produced 8.35 1 H2/tool glucose utilized. The role of nitrogenase system in H2 generation was evident by derepressed nitrogenase activity (0.46 nmoles C2H4 produced/mg protein/h) under defined in vitro conditions. For maximum expression of the activity, the cells required preactivation under anaerobic conditions by incubating at 40 ~ for 20-24h with 0.2% glucose in the culture medium. At an 0 2 level of more than 0.25%, the acetylene reduction activity decreased significantly and could not be detected at a level of 20%. Nitrogenase activity development was higher at acetylene: inoculum ratio between 4.2 6.25. H2 evolution was lower when the mixed cells were incubated under an atmosphere of 10% C2H2 and 5%CO gas. This decrease in H2 evolution was also evident at 2.5 - 6.5 mM NaNO3 and KNO3 concentrations in the liquid culture medium thus establishing more than 50% H2 evolution through nitrogenase.

Key words: Acetylene reduction, Hydrogen, Mixed culture, Nitrogenase, Nitrogenase inhibitors ~TRODUCTION Hydrogen (H2) is considered to be an ideal and pollution free fuel of the future (1, 2). Since the discovery of biological hydrogen production by green algae (3), its production by both phototrophic and chemotrophic prokaryotes have been investigated by many authors (4, 5, 6). Increased and continuous hydrogen production by these organisms has been of greater interest (4, 7, 8). H2 metabolism in most H2 evolving prokaryotes involve hydrogenase and nitrogenase enzyme complexes (4). Nitrogenase, in almost all prokaryotes evolves H2 to different extents while converting molecular nitrogen to ammonia, the process termed as

* To whom all correspondence should be addressed at the present address: Department of Biotechnology, Centerfor Chemistry and Chemical Engineering, Lund University, P.O. Box 124, Lund, S-22100, Sweden. Fax: +46-46-2224713. [039-9712/98/080245-09505.00/0 245

Copyright 9 1998 by Academic Press Austr~tlia All rights of reproduction in any fi~rm reserved.

Vol. 45, No. 2, 1998

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

biological nitrogen fixation (9, 10). In fermentative H2 production the nature of H2 metabolism known so far is through E. coli and Clostridia fermentative pathways. H2 evolution in these processes is in general treated as a means to release excess protons (4, 5, 6). Involvement of nitrogenase in fermentative H2 production is not clearly known. Nitrogenase activity in such systems is routinely measured by the ability of the enzyme to reduce acetylene to ethylene. The assay is being frequently used for free cells or for the cells in symbiotic association (11, 12 ). The estimation of nitrogenase activity in mixed microbial cultures under anaerobic conditions by acetylene reduction assay has paid little attention. In the present study, we describe the effect of some physiological factors on the development of nitrogenase activity in mixed microbial culture. This allows an investigation of the nitrogenase dependent fermentative H2 evolution in the mixed culture by using enzyme inhibitors. M A T E R I A L S AND M E T H O D S

Organism. Mixed microbial population was obtained from fresh cattle dung. One liter of 3% cattle dung slurry was passed through fine muslin cloth. The filtrate was treated for enrichment of H2 producing organisms (13- 15). The mixture was allowed to stand for lh to remove heavy matter. The supernatant recovered was centrifuged at 2,000 rpm. The pellet was discarded and the supernatant was again centrifuged at 10,000 rpm. The enriched cell pellet so obtained was resuspended in 50 ml of the supernatant and used for further experimentation.

Fermentative H2 evolution. 150 ml of 3% (w/v) glucose solution was inoculated with 5 ml of mixed culture and incubated in 300 ml aspirator bottle (9). pH was adjusted to 7.0 daily with 1.0 M NaOH and the mixture was flushed with N 2 and incubated at 40~ Incubation was continued until H2 evolution ceased.

Nitrogenase Activity (Acetylene reduction). Acetylene reducing activity of the mixed culture was determined in the incubation assemblies (Figure 1) having total void volume of 65 - 70 ml. The assemblies were made air tight and provided with latex tubes for injecting gases. 3 ml cells (60 mg dry wt or 4.3 mg protein/ml) were inoculated in 22 ml of 0.2% sterile glucose solution and placed in the incubation assembly. The mixture was neutralized and flushed with argon gas to create anaerobic conditions. The cells were activated by incubating at 40~ for 20 - 24 h till H2 production rate was maximum. The cell mixture was again neutralized and whole assembly was flushed with argon gas to create an inert atmosphere. At this stage 0.1 atm (10% v/v) of acetylene environment was created by injecting 4.5 ml of acetylene gas in the assembly. The mixture was incubated at 40~ till 70 h and during this incubation the gas samples were withdrawn at regular intervals for ethylene determination. Derepression of the nitrogenase activity was checked under different conditions: a) The assay was conducted without activating the cells with glucose. The cells were just incubated with sterile distilled water and the effect of.pre incubation for 20 - 24 h was studied. b) Effect of O2 was studied by varying 02 concentrations from 0.25 - 20% (v/v). The cells were first activated and preincubated as above in the incubation assemblies. Different volumes of air were added in the assemblies to achieve different O2 concentrations. At this point assemblies were also injected with 10% acetylene

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atmosphere. The assemblies were incubated for 20 h at 40 ~ and the gas samples were withdrawn at 12 and 20 h for ethylene determination. The effect of 0 2 was checked on shake and stationary culture. c) Acetylene reduction was also conducted by varying the Inoculum : acetylene ratio. Cell volumes from 5 - 35 ml was taken in the incubation assemblies and acetylene from 3.1 - 6.0 ml was injected to create the appropriate 10% v/v acetylene atmosphere. The assemblies were incubated for 20 h at 40 ~ and the gas samples were withdrawn at 12 and 20 h for ethylene determination.

Hydrogen production by Nitrogenase. The effect of various nitrogenase inhibitors on hydrogen production was studied. 3% glucose solution was inoculated with enriched mixed culture of H2 producers at neutral pH. 30 ml of this mixture was placed in individual incubation assemblies (Figure 1). The assemblies were flushed with argon. CO gas concentration of 2%, and 5% were created in the assemblies by injecting respective volumes of the gas. The mixtures were incubated at 40~ in the stationary culture and on shaking (110 rpm) for 24h. Control experiment was run under similar conditions without CO. The effect of C2H2 on H2 production at 10% v/v concentration was also studied in the similar manner. The gases were collected and analysed for H2 and CO2. Similar effects were also tested in experiments by adding different concentrations of KNO3 (2.5 - 8.0 mM) and NaNO3 (2.5 - 5.0 mM) in culture medium. The cell mixtures were incubated for 24 h at 40 ~ All the treatments were run in triplicates and experiments repeated thrice.

Gas collection and analysis. Gases (H2 and CO2) were collected over water (pH 2.0) in inverted measuring cylinder (Figure 1) and the volumes calculated at 25~ Gas analysis was done on gas chromatograph using standard GC procedures. For analysing H2 and CO2 molecular sieve and porapak-Q columns were used respectively at ambient temperature on thermal conductivity detector. Acetylene and ethylene were analysed on Porapak-T column using flame ionization detector.

Determination of whole cell protein. The cell extract was produced by the method of Herbert et al (16) and protein concentration was determined by Lowry's method (17). Glucose estimation. Glucose was estimated by dinitrosalicylic acid method as described in the Worthington Manual (18). RESULTS AND DISCUSSION Batch culture fermentation of glucose to H2 by mixed microbial culture leads to the generation of 15.2 1 of gaseous mixture (H2 and CO2) per mol of glucose utilized. H2 constituted 55% of the total gas produced thus evolving 8.35 1 H2/mol glucose utilized. The bioconversion efficiency of glucose was about 100%. The capacity of the culture to generate substantial amount of H2 under anaerobic conditions leads to the curiosity of looking for the nitrogenase system in the culture.

Nitrogenase activity under defined conditions Acetylene reduction (nitrogenase) activity of the free living cells of mixed culture was determined under specific conditions like preincubation, 0 2 exposure and change in inoculum size. Maximum activity of 0.46 nmoles C2H4 produced/mg protein/h was 247

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H

W-

Figure 1. Incubation assembly for hydrogen production and nitrogenase estimation. A: Incubation mixture containing mixed culture and glucose solution. I: inlet for adjusting pH with 1N NaOH and flushing argon gas; I': Inlet for injecting gas inhibitors and taking gas samples for analysis; W: Water at pH 2.0 and H: Measuring cylinder for collecting gases.

expressed during 12 - 20 h of incubation period (Table 1). 0 2 levels of more than 0.25% were inhibitory for acetylene reduction and H2 evolution under both stationary and shake culture conditions (Figure 2 & 3). In shake culture conditions, 20% 0 2 led to complete loss of nitrogenase activity as compared to 60% inhibition in stationary culture. This resulted from greater exposure of cells to 0 2 toxicity. The decline in nitrogenase activity was also expressed by decrease in the H2 evolution capacity as the 0 2 concentration increased (Figure 3). At 5% 0 2 concentration (in shake culture), although the nitrogenase activity decreased by only 44%, but the decrease in hydrogen generation was quite high (72%). This greater decrease in H2 generation may have resulted because of the significant inactivation of hydrogenase enzyme also besides nitrogenase (2) and partly by the consumption of H2 in the presence of 02. In contrast, the observations recorded by other workers showed mixed effect of 0 2 exposure based upon the culture (19, 20). Table 2 describes the effect on nitrogenase activity by varying acetylene : inoculum ratio in the assay system. The optimum activity was found to be between 4.2 - 6.25 ratio. Preactivation of the culture in presence of 0.2% glucose solution by incubating at 40~ for 20 - 24 h was observed to be an essential requirement for the development of nitrogenase activity (Table 3). Without incubating or in absence of glucose the activity decreased by 63 - 77%.

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Table 1. Nitrogenase activity of mixed microbial culture at different incubation periods. Incubation time #

Incubation phase 82

(h)

Nitrogenase activity * X+SD

(h)

Nitrogenase activitry * X+SD

0

0.0 + 0.0

0-2

0.069 + 0.01

2

0.069 + 0.01

2-6

0.280 + 0.04

6

0.210 + 0.03

6 -12

0.433 + 0.02

12

0.322 + 0.02

12 -20

0.460 + 0.03

20

0.376 + 0.04

20 - 70

0.088 + 0.03

70

0.170 + 0.03

* Nitrogenase activity is determined by acetylene reduction assay and is expressed in nmoles C2H4 produced/mg protein/h. # Incubation time represents the total time of incubation from the beginning and nitrogenase activity was calculated by measuring the total C2H4 produced upto that incubation time and finally expressed per hour. 82Incubation phase represents the time period between two time intervals at which nitrogenase activity was measured. Nitrogenase activity in this case is calculated by measuring the C2H4 produced during specified incubation phase and finally expressed per hour. X + SD = Mean + Standard deviation. Values are based on six experiments.

Evolution of H2 through nitrogenase and hydrogenase Table 4 describes the effect of nitrogenase and hydrogenase enzyme inhibitor on the evolution of H2. At an optimum concentration of 5% CO level in the gas phase, H2 production was inhibited by 31% in the stationary cultures. The shake culture showed more pronounced effect because of the better exposure of the cells to enzyme inhibitor. Similar effects were also observed when 10% C2H2 were used in the gas phase. However, in combination of the C2H2 and CO almost total inhibition of H2 evolution occurred in shake culture. These effects were quite similar to those observed by various other workers using different cultures (21 - 25). Complete inhibition of H2 evolution by

Rhodobacter sphaeroides in the presence of 10% C2H2 indicates the presence of nitrogenase mediated hydrogen evolution (21). It has also been suggested that acetylene inhibits conventional hydrogenase activity and thus prevent the uptake of H2 formed by nitrogenase in the presence of 5% CO (22). CO also blocks acetylene reduction activity but not the hydrogen evolving function of nitrogenase (22, 23). To assess the reversibility of the effect of inhibitor, it was observed during the course of our studies,

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0.5

F-.-

0.4

I-- L_ m E