Nitrogenase

Among various technologies for hydrogen production, the use of oxygenic natural photosynthesis has a great potential as can use clean and cheap sources-water and solar energy. In oxygenic photosynthetic microorganisms electrons and protons produced from water and redirected by the photosynthetic electron-transport chain via ferredoxin to the hydrogen-producing enzymes hydrogenase or nitrogenase. By these enzymes, e − and H + recombine and form molecular hydrogen. Hydrogenase activity can be very high but is extremely sensitive to the photosynthetically evolved O 2 that leads to reduced and unstable H 2 production. However, presently, several approaches are developed to improve the energetic efficiency to generate H 2 . This review examines the main available pathways to improve the photosynthetic H 2 production.

Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H 2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H 2 production. The growth, H 2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H 2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H 2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H 2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Azotobacter chroococcum (A101) was examined for some biological activities such as nitrogenase, phosphatase, potassium solubilization, and production of some plant hormones such as indole acetic acid, gibberellic acid, and cytokinin. Six different formulations were prepared, using different carrier materials namely; peat moss, mixture of peat moss plus vermiculite 1:2 (w/w), wheat bran, rice husk, clay, and sodium alginate. Each carrier material was packed using polyethylene pages, and then divided into three groups. The first group was sterilized by autoclaving at 121°C for 20 min, and the second one was sterilized by gamma irradiation at a dose rate of 4.0 kGy for 1 h. However, the third group was left without sterilization. Half of the inoculated polyethylene bags, containing the tested formulations either sterilized by autoclaving or gamma irradiation, were incubated at 8°C and the other bags were incubated at 30°C for 6 months. The nonsterilized bags were incubated under the same condition but only for 3 months. For testing the survival of Az. chroococcum (A101), the prepared formulation samples were taken every month during the storage period. Nitrogenase activity was evaluated in the prepared formulations which exerted survival cells equal to or more than 10 8 CFU/ml after 6 months of storage period. Results revealed that non-sterile formulations exerted high numbers of total fungi and bacteria along the storage period; however, Az. chroococcum (A101) numbers were decreased over incubation time. No contam-

An attempt was made in the current study for
the isolation of endophytic bacteria having nitrogen fixing
potential from the roots of young leguminous plant, Parkia
roxburghii (Tree bean). Four different spots were selected
from Imphal east district and the root samples are collected
from four different young plants. A total of five (5) bacterial
isolates were isolated using nitrogen free Yeast Extract
Mannitol Agar medium from the roots of the plant.
Preliminary physiological tests and biochemical tests were
being carried out for the isolates for assessment of their
nature. Nif gene amplification was carried out to determine
the nitrogenase activity of the bacterial isolates.
Incorporation of 16S rDNA analysis for three selected
bacterial isolates positioned themselves as highly similar to
be Pseudomonas hibiscicola, Bacillus subtilis and
Agrobacterium fabrum.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in fieldgrown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

Acetobacter diazotrophicus, an acid-tolerant endophytic bacterium which grows best on a sucrose-rich medium. In a series of experiments, aseptically-grown sugar cane plantlets were rooted in a liquid medium and inoculated with A. diazotrophicus originally isolated from field-grown sugar cane. After 4, 7, 9, and 15 d, plants were examined under light, scanning and transmission electron microscopes and the presence of A. diazotrophicus on and within plant tissues was confirmed by immunogold labelling. By 15 d, external bacterial colonization was seen on roots and lower stems, particularly at cavities in lateral root junctions. The loose cells of the root cap at root tips were a site of entry of the bacteria into root tissues. Both at lateral root junctions and root tips, bacteria were also seen in enlarged, apparently intact, epidermal cells. After 15 d, bacteria were present in xylem vessels at the base of the stem, many connected via mucus to spiral secondary thickening. There was no obvious pathogenic reaction to the bacteria within the xylem. From these observations, it is proposed that, under experimental conditions, A. diazotrophicus firstly colonized the root and lower stem epidermal surfaces and then used root tips and lateral root junctions to enter the sugar cane plant where it was distributed around the plant in the transpiration stream. It is further suggested that the xylem vessels in the dense shoots of mature plants are also a possible site of N 2 -fixation by diazotrophs as they provide the low pO 2 and energy as sucrose necessary for nitrogenase activity.