Hydrogen metabolism of mutant forms of Anabaena variabilis in continuous cultures and under nutritional stress
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Nitrogenase activity and H2 evolution were studied in two mutants of the cyanobacterium Anabaena variabilis ATCC 29413 which are impaired in molecular H2-related metabolism. Evidence was obtained that mutants deficient in uptake and reversible hydrogenases were suitable for biotechnological research on H2 production. H2 production by the mutant PK84 in continuous cultures was 4.3 times higher compared to the wild-type. Enhancement in H2 evolution by all the cultures under N2 (1.8–1.9 times) and CO2 starvation (1.4–1.5 times) was observed.
H2 production using biological materials is one of the goals of renewable energy technology. H2 evolution is a conservative process inherent in most N2-fixing microorganisms and involves the two enzymes – nitrogenase and reversible hydrogenase . Cyanobacteria are suited for biotechnological H2 production as they are the only photoautotrophic N2-fixers capable of producing molecular H2 with H2O as the electron source and they are stable towards various stress factors [2–7].
Several species of cyanobacteria are presently being used in H2 photoproduction research and development, Anabaena variabilis among them . In addition to simple selection of the strains, genetic methods are being applied to create mutants with altered nitrogenase or hydrogenase systems for enhanced H2 production capacity. Furthermore, physiological manipulation can be used for optimising H2 production .
Nitrogenase activity and H2 production in three strains of A. variabilis (the wild-type and two mutant forms) are presented here using continuous cultures during the exponential growth phase in a steady-state mode. This phase is characterised by the highest activities of the enzymes involved in H2 metabolism and is of interest for the practical cultivation of various cyanobacterial species [5, 6, 8, 10, 11]. In the present research CO2 or N2 starvation was used to establish the possibility of regulation of the H2 metabolism of A. variabilis both for basic knowledge and as a background for applied biotechnology.
2Materials and methods
Three strains of the cyanobacterium A. variabilis ATCC 29413 were used: the wild-type (the initial form) and two new chemically generated mutant forms, PK84 and PK17R, provided by Professor S.V. Shestakov and Dr. L.A. Mikheeva of Moscow State University, Department of Genetics. The mutations affected regulation of the enzymes of H2 metabolism, thereby enhancing yields of molecular H2 evolution. Reversible hydrogenase activity was impaired in the mutant PK84, and both forms were deficient in uptake hydrogenase . However, physiological parameters such as nitrogenase activity, growth rates, heterocyst frequency, etc. were similar in all three forms .
Continuous growth of the cyanobacteria was carried out in thermostated 350 ml glass bioreactors equipped with a pH-control system and permanently illuminated with daylight fluorescent lamps at a limiting light intensity of 90 μE m−2 s−1. The dilution rate was 0.03 h−1. The gas mixture contained 25% N2, 2% CO2, and 73% Ar. Total gas flow was 250 ml min−1. Metabolic stress conditions were achieved by lowering the N2 content to 5% or switching off the CO2 supply. Modified nitrogen-free Allen and Arnon medium  was used for N2-fixing growth. A pH of 7.5 was maintained automatically by addition of NaOH.
2.3Heterocyst frequency, assays of chlorophyll, protein and enzyme activities
Heterocysts were counted visually under a microscope taking not less than 500 cells at a time. The frequency in Tables 1–3 is given in percent related to the total number of cells counted.
Table 1. Growth parameters and enzymatic activity of continuous cultures of the wild-type (W.T.) and two mutants (PK84 and PK17R) of Anabaena variabilis (standard deviation did not exceed 10% of the values)
Table 2. Maximal effect of nitrogen starvation (5% N2) upon growth parameters and enzymatic activity of the wild-type (W.T.) and two mutants (PK84 and PK17R) of Anabaena variabilis measured at 48 h after starting the treatment (standard deviation did not exceed 10% of the values)
Table 3. Maximal effect of carbon starvation (0% CO2) upon growth parameters and enzymatic activity of continuous cultures of wild-type (W.T.) and two mutants (PK84 and PK17R) of Anabaena variabilis measured at 24 h after starting the treatment (standard deviation did not exceed 10% of the values)
Chlorophyll content was determined in methanol extracts prepared by incubation of cells in 90% methanol for 3 min at 75°C . An optical density (OD665) of 1 corresponded to 13.4 μg Chl a ml−1 of the culture.
Protein content was determined by the method of Bradford . One unit of optical density (OD595) corresponded to 1.3 μg of protein ml−1. All the reagents were the highest grades commercially available.
Enzyme activities in whole cells were determined by assaying H2 photoproduction and C2H2 reduction. The gases were monitored by gas chromatography (Hewlett Packard 5890) after incubation of the samples in glass vials at 30°C for 30 min under daylight lamp fluorescent illumination (140 μE s−1 m−2). The gas phase for H2 evolution assay contained only Ar, whereas C2H2 was added at 10% (v/v) for the C2H2 reduction assay and the C2H4 produced by nitrogenase was detected.
3Results and discussion
Continuous cultivation is often used in biotechnology for maintaining cultures in the exponential phase of growth, obtaining higher yields of biomass and higher enzyme activity. We have chosen this method since the maximal activity of nitrogenase occurs during the exponential growth phase , and it is known that hydrogenase activities in the mutants increased at later stages of growth . The wild-type and two mutants were maintained for not less than three doubling times in chemostats as light-limited continuous cultures in order to establish equivalent conditions for the cultures before the treatment with low levels of N2 and CO2.
3.1Growth parameters in continuous cultures
The data on biomass accumulation (dry weight), protein content, chlorophyll concentration and heterocyst frequency showed that both mutants did not exhibit any noticeable changes in their physiological properties compared to the wild-type while growing as continuous cultures under the same conditions (Table 1). The results were in agreement with the preliminary data obtained for the strains in batch cultures  and confirmed the viability of the mutants.
3.2.1Nitrogenase activity of the cells during continuous cultivation
Two aspects of the reducing function of nitrogenase were studied – the reduction of C2H2 (as a substrate equivalent to molecular N2) to C2H4 and molecular H2 evolution.
The data showed clear differences between the three strains of A. variabilis in the quantitative yield of the gases produced by nitrogenase in continuous cultures (Table 1). Both mutant forms showed higher rates of H2 evolution than the wild-type. The most pronounced differences were obtained between the mutant PK84 and the wild-type: this mutant evolved 4.3 times more H2 than the wild-type (Table 1), whereas the amounts of H2 evolved by the PK17R mutant strain differed from the wild-type by 1.4 times (Table 1).
In the case of C2H2 reduction (Table 1) the differences between the enzyme activities in different strains during continuous cultivation were less pronounced, although the nitrogenase activity in the mutants was still slightly higher than in the wild-type (about 1.2 times). The impaired H2 uptake activity of hydrogenases  is thus the probable cause of the higher H2 evolution rates observed in the mutants.
3.2.2The influence of nitrogen and carbon deficiency
Stress factors (including starvation by the main nutrients – carbon and nitrogen) can lead to a temporary increase of the activities of enzymes involved in the main metabolic pathways. This hypothesis was tested by placing the cyanobacteria under nutritional stress of N2 and CO2 starvation.
After stable growth in continuous cultures had been reached the N2 content of the gas phase was decreased from 25% to 5%. This decrease in N2 supply produced its effect after 24 h with a maximal response after 48 h (Table 2). The nitrogenase activity increased rapidly with the patterns similar for each strain. H2 production was 1.9 times higher in the wild-type and about 1.8 times higher in PK84 and in PK17R. The patterns of C2H2 reduction were also quite similar: 2.2 times increase in the wild-type, and about 1.8 times increase in both mutants (Table 2 versus Table 1).
All the other features – dry weight, protein content, chlorophyll concentration – decreased considerably 48 h after the beginning of the N2 starvation treatment (Table 2 versus Table 1). All these changes were reversible upon the addition of N2 to the gas phase and the cultures could restore all the measured parameters to the levels shown in Table 1.
These results show the possibility of the regulation of H2 metabolism in cyanobacteria by N2 supply in order to improve the efficiency of light energy conversion for H2 production.
The procedure for changing the growth conditions in order to study carbon starvation was similar to that described for N2. The carbon stress effect reached its maximum within 24 h after stopping the CO2 supply, but the rate of H2 evolution and C2H2 reduction (Table 3) was somewhat lower than that achieved after 48 h of N2 deficiency (Table 2). With decreased CO2 the H2 evolution activity increased about 1.5 times in both the wild-type and the mutants. The C2H2 reduction activity of the nitrogenase increased also, but to a lesser extent than that observed in the absence of N2 (a 1.5–1.7 times increase in the wild-type and in the mutants). CO2 depletion effects after 24 h resulted in an irreversible decrease in the other measured physiological parameters (Table 3 versus Table 1).
A comparison of the physiological effects of carbon and nitrogen stress on H2 metabolism in three strains of Anabaena variabilis indicated that the PK84 mutant is the most appropriate form for use in photobioreactors designed for H2 production. Deficiencies in N2 and CO2 resulted in substantial increases in H2 evolution activity. Continuous cultures with optimal nitrogenase activity can be established for long-term H2 production.
The project was supported by RITE (Japan), INTAS (Brussels) and the Royal Society (UK).