Selection of starter cultures for the production of kinema,a fermented soybean food of the Himalaya

Kinema, a traditional fermented soybean food, serves as a low-cost source of protein in the diet of the people of the Himalaya. The traditional method of kinema preparation results in a product with inconsistent quality. Forty five strains of spore-forming bacteria were isolated from nine samples of kinema collected from markets in the Darjeeling hills and Sikkim in India. Of these, 10 strains, identified as Bacillus subtilis (Ehrenberg) Cohn, were selected as possible starter cultures on the basis of enzyme activities and the production of slimy material. Protease activity (U/ml), -amylase activity (U/ml) and relative viscosity of the selected strains were 6.5 to 81.5, 0.1 to 9.3, and 1.1 to 20.1, respectively. Kinema produced by these strains showed nitrogen contents (expressed as a percentage of the total nitrogen content) of: water-soluble N, 48.4 to 76.5; TCA-soluble N, 16.0 to 27.6; formal N, 5.0 to 12.5; ammonia-N, 4.4 to 7.8%. Reducing sugar was 1.1 to 2.0% of the wet weight and relative viscosity was from 2.0 to 35.2. Correlation matrices of the biochemical parameters and sensory attributes of the kinema produced by these B. subtilis strains were statistically analysed. Strains KK-2:B10 and GK-2:B10 of B. subtilis were the best starter cultures for improved kinema production.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

Summary Nitrogen fixation was induced in a strain of cowpea rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C₂H₂ reduction, was maximized at an O₂ concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH₄ ⁺ and NO₃ ^- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C₂H₂ in association with tobacco cells; the highest levels of activity were found among cowpea strains.     

Nitrogen fixation by Rhizobium in pure cultures.

Strains of Rhizobium trifolii and Rhizobium meliloti were tested for their asymbiotic nitrogen fixation ability. From among ten tested strains two R. trifolii and one R. meliloti expressed nitrogenase activity within the range of 1.3--9.3 nM C2H4/h/mg protein. Asymbiotic nitrogen fixation was affected by the composition of the medium.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

The aerobic hydrogen-oxidizing bacterium Alcaligenes latus represented by three strains was found to be able to grow with dinitrogen as the sole nitrogen source: The doubling time of total (Kjeldahl) nitrogen during growth on glucose at 30°C under an atmosphere containing 2% (v/v) oxygen in dinitrogen amounted to 39 h, while that in the presence of ammonium was 3 h. Nitrogen fixation did apparently not occur under air. During diazotrophic growth the cells accumulated up to 75% (w/dry weight) poly--hydroxybutyric acid. The efficiency of nitrogen fixation varied between 10 and 15 mg N per g glucose utilized. The specific nitrogenase activity measured in the acetylene reduction assay amounted to 5–17 nmol C₂H₄ formed per min and mg protein.     

Effects of phosphorus supply on in vitro growth and phosphatase activity of Frankia isolates from Casuarina

An in vitro experiment was conducted to investigate the effects of different sources and levels of P supply on growth, viability and phosphatase activity of three tropical Frankia strains isolated from Casuarina. P concentration for optimum growth was between 0.1 and 10.0 M in the absence of external combined nitrogen. Specific viability was not influenced by P supply. Morphological features of Frankia, such as hyphal length and vesicle numbers, were found to largely mirror growth. Phosphatase activity was detected in all three Frankia strains and was highest when P was omitted from the culture solution. There were more than 10-fold differences between the Frankia strains in the level of phosphatase activities shown. This study suggested that soils low in P are unlikely to restrict micro-symbiont growth activity.     

Regulatory aspects of inorganic nitrogen metabolism in the Rhodospirillaceae

Regulatory aspects of the assimilation of inorganic nitrogen compounds (ammonia, nitrate, nitrogen) were studied in 12 strains belonging to the Rhodospirillaceae. All strains possessed an ammonium transport system, as demonstrated by ¹⁴C-methylammonium uptake. This uptake showed saturation kinetics (K _m between 50–150 M), and was competitively inhibited by ammonium (K _i between 5–18 M). The ammonium transport systems were repressed by ammonium in the growth medium. The nitrogenase activity of all strains was reversibly inhibited by ammonium (switch-off). This effect was not shown under nitrogen starvation conditions with the exception of some strains of Rhodopseudomonas capsulata, the nitrogenase of which was always susceptible to switch-off by ammonium. Assimilation of nitrate was confined to some strains of Rhodopseudomonas capsulata.Abbreviations ATCC American Type culture Collection, Rockville, MD, USA - DSM Deutsche Sammlung von Mikroorganismen, Göttingen, FRG     

Effect of plant and artificial aeration on solids accumulation and biological activities in constructed wetlands

In constructed wetlands, solids accumulation may have two consequences with opposing effects on treatment efficiency: it decreases the longevity by reducing void space and it enhances biological activity by favoring biofilm development. The goal of our study was to estimate the effect of plants (presence and species) and artificial aeration on solids accumulation (volatile and inorganic). The horizontal and vertical distribution of solids was sampled using solids traps in 12 constructed wetland mesocosms (5 years old). Microbial density and activity were estimated in the biological fraction of the sampled solids. The effect of plant presence reduced accumulated solids by 26% and sulphide content by 50% sulphide content. There was more solids accumulation in Typha angustifolia units than in Phragmites australis. Also, T. angustifolia generated more biological activities at the surface and close to the inlet while conditions were more homogeneous throughout P. australis units. Aeration (1) stimulated biofilm development at the inlet of planted beds, (2) seemed to reduce mineral matter accumulation and (3) generated the same pattern of activities in planted beds enabling to reach a total nitrogen removal rate of up to 0.65 g N m^?2 d^?1.     

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of litter nitrogen on decomposition and microbial biomass inSpartina alterniflora

The effect on decomposition of 4 different levels of nitrogen in aerial tissue ofSpartina alterniflora, collected at the end of its growing season litter, was studied in laboratory percolators for 56 days at 20C. The CO₂ evolution and the release of organic nitrogen and organic carbon were monitored. From these data, the ash-free dry weight (AFDW), nitrogen (N) content, and carbonnitrogen (C/N) ratio were calculated at various times during decomposition. Fungal biomass, bacterial biomass, and the relative autoradiographic activity of bacteria were measured at the end of the study. Decomposition was significantly affected by the nitrogen content of the litter. A 55% increase in plant N increased overall weight loss and k by 50% and 40%, respectively. Furthermore, k (calculated from time course weight loss data) responded linearly to the 4 different levels of nitrogen inSpartina tissue. Fungi appear to dominate the microbial community. At the end of the experiment, fungal biomass was between 2.23 and 3.08% of the AFDW, and was calculated to contain 12 to 22% of the nitrogen in the litter. Bacterial biomass was 1/10 of the fungal biomass, and 12–17% of the bacteria were active. The total microbial biomass was not affected by increased plant nitrogen. In the course of decomposition, the organic nitrogen and carbon were highest in the effluent water in all treatments during the first 8 days. The respiration rate (CO₂ evolution) first increased to a maximum at day 18 and then decreased to a constant rate (1–2 mg C/day/g detritus). Respiration was highest in the high N litter. The C/N ratio in all treatments increased from the start to day 8, then decreased to day 20. In low N litter, C/N then increased again as a result of increased total organic nitrogen (TON) loss relative to carbon mineralization. In the high N, this was reversed.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Impact of nitrogen and drought on phycobiliprotein content in terrestrial cyanobacterial strains

Cyanobacteria are highly adaptable microorganisms, characterized by an ability to survive in different environments with unfavorable conditions. In order to determine the content and composition of phycobiliproteins in cyanobacterial strains, depending on environmental factors, such as drought, darkness, and lack of nutrients in the substrate, the quantity of these pigments was investigated in 21 cyanobacterial strains. The study was conducted with terrestrial, filamentous, N₂-fixing cyanobacterial strains, which belong to Nostoc and Anabaena genera, isolated from different soil types in the Vojvodina region (Serbia). All strains were cultivated in nitrogen-free medium, medium with nitrate concentration of 2 g?L^?1 in the presence of light, and in drought conditions (water activity less than 0.5 %), on inorganic substrate, in the dark. Statistically significant differences of phycocyanin and allophycocyanin contents were found in the case of strains cultivated in nitrogen-free medium compared to strains grown in the medium containing nitrogen. There were no statistically significant differences in phycoerythrin and total phycobilin contents between strains grown in media with and without nitrogen. The results suggest that nitrogen availability affects composition of phycobiliproteins in the tested strains without affecting the total phycobiliprotein content, which implies maintenance of balanced pigment abundance as cyanobacterial response to nitrogen source. Significantly lower concentrations of the studied pigments, which varied from 0.8 to 16.9 μg mg^?1 dry weight, were detected in strains exposed to dry and dark conditions. These data indicate the presence of preserved phycobiliproteins after a period of 10 years of cyanobacterial exposure to drought and darkness, which can be of great importance for the study on their stability under unfavorable environmental conditions.     

Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO₃ combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO₃, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Growth of selected fungi on leaf-protein supernatant

Summary Some 19 strains ofAspergillus niger,A. oryzae, andPaccilomyces spp. are tested for their ability to grow on the supernatant remaining after the expressed juice from sugar beet tops and meadow grass has been heat-treated to precipitate crude leaf protein, and supplemented as required by glucose or ammonium sulphate. With effective strains ofA. niger,A. oryzae,P. elegans orP. variotii and an optimized carbohydrate/nitrogen ratio, over 70% of the organic content of the supernatant is rapidly converted into mycelial biomass of high protein content.     

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

Diversity and bioactivities of nostocacean cyanobacteria isolated from paddy soil in Vietnam

Nostocacean cyanobacteria are one of the important components of paddy fields due to their ability to fix atmospheric nitrogen and supply phytohormones for crop growth. In this study, 13 Nostoc strains isolated from paddy soils in Vietnam were classified using a polyphasic approach. The results showed a high diversity of the isolated strains that represented seven morphotypes corresponding to five genotypes, with 16S rRNA gene sequence similarity values ranging between 94.97–99.78% compared to the available sequences from GenBank. Bioassay assessment revealed that 11 out of 13 strains possessed antibacterial activities, three of which exhibited cytotoxic activities on MCF7 and HCT116 cells with an IC₅₀ ranging from 47.8 μg mL^?1 to 232.0 μg mL^?1. Interestingly, strains with identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activity profiles.     

Studies on the respiration of efficient and inefficient strains of Rhizobium

Summary In a pot culture experiment using sterilised soil, growth and nitrogen content of Berseem (Trifolium alexandrinum) inoculated with 15 strains of Rhizobium trifolii and of pea (Pisum sativum) inoculated with 10 strains of Rhizobium leguminosarum were found to vary considerably depending on the strains of the respective Rhizobium used. Out of the 15 strains of Rhizobium trifolii, 6 strains were found to be highly efficient (increasing the nitrogen content by more than 70 per cent over the control-uninoculated) and the rest as either moderately efficient (increase in N by 30–50 per cent) or inefficient (increase in N by 4 to 20 per cent) strains. Similarly, out of the 10 strains of Rhizobium leguminosarum 5 strains were found to be highly efficient, 1 moderately efficient and the rest were more or less inefficient strains.The respiration rate (l O₂ consumed per hour per mg dry cells) of efficient and inefficient strains of Rhizobium trifolii and Rhizobium leguminosarum in glucose, maltose and mannitol substrate did not bear any relation to their efficiency. However, the stimulation of the respiratory rate of the Rhizobium strains due to the addition of glycine to the glucose substrate was found to be significantly more in case of efficient strains of Rhizobium trifolii and Rhizobium leguminosarum than those of the inefficient ones.The data presented in the paper is taken from the thesis submitted by the Senior author, to the P. G. School, IARI, N. Delhi, in 1968, for Ph. D. degree.