On the control mechanisms of the nitrite level in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells: the mathematical model

Background

Due to a high toxicity of nitrite and its metabolites, it is of high interest to study mechanisms underlying the low NO₂ level maintenance in the cell. During anaerobic growth of Escherichia coli the main nitrite-reducing enzymes are NrfA and NirB nitrite reductases. NrfA reductase is localized in the cell periplasm and uses NO₂ as an electron acceptor to create a proton gradient; NirB reductase is restricted to the cytoplasm and metabolizes excessive nitrite inside the cell, the uptake of which is mediated by the transporter protein NirC. While it is known that these three systems, periplasmic, cytoplasmic and transport, determine nitrite uptake and assimilation in the cell as well as its excretion, little is known about their co-ordination.

Results

Using a mathematical model describing the nitrite utilization in E. coli cells cultured in a flow chemostat, the role of enzymes involved in nitrite metabolism and transport in controlling nitrite intracellular levels was investigated. It was demonstrated that the model adapted to the experimental data on expression of nrfA and nirB genes encoding NrfA and NirB nitrite reductases, can describe nitrite accumulation kinetics in the chemostat in the millimolar range of added substrate concentrations without any additional assumptions. According to the model, in this range, low intracellular nitrite level, weakly dependent on its concentration in the growth media, is maintained (mcM). It is not sufficient to consider molecular-genetic mechanisms of NrfA reductase activity regulation to describe the nitrite accumulation dynamics in the chemostat in the micromolar range (≤1 mM) of added nitrite concentrations. Analysis of different hypotheses has shown that the mechanism of local enzyme concentration change due to membrane potential-induced diffusion from the cytoplasm to the periplasm at low nitrite levels is sufficient to explain the nitrite accumulation dynamics in the chemostat.

Conclusions

At nitrite concentrations in the media more than 2 mM, the model adapted to the experimental data on nitrite utilization dynamics in E. coli cells cultured in the flow chemostat demonstrates the largest contribution of genetic mechanisms involved in nrf and nir operons activity regulation to the control of nitrite intracellular levels. The model predicts a significant contribution of the membrane potential to the periplasmic NrfA nitrite reductase activity regulation and nitrite utilization dynamics at substrate concentrations ≤1 mM.

     

A study of the nitrate and nitrite discharge from the mutant cells of <Emphasis Type="Italic">Neurospora crassa</Emphasis> lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Engineering of an L-arabinose metabolic pathway in <Emphasis Type="Italic">Rhodococcus jostii</Emphasis> RHA1 for biofuel production

The oleaginous bacterium, Rhodococcus jostii RHA1 has attracted considerable attention due to its capability to accumulate significant levels of triacylglycerol as renewable hydrocarbon. To enable the strain to utilize arabinose derived from lignocellulosic biomass, the metabolic pathway of L-arabinose utilization was introduced into R. jostii RHA1 by heterogenous expression of the operon, araBAD from Escherichia coli. The results showed that recombinant bearing araBAD could grow on L-arabinose as the sole carbon source, and additional expression of araFGH encoding the arabinose transporter from E. coli could improve the cell biomass yield from high contents of arabinose. We further increased the content of lipid produced from arabinose in the recombinants from 47.9 to 56.8 % of the cell dry weight (CDW) by overexpression of a gene, atf1 encoding a diglyceride acyltransferase from R. opacus PD630. This work demonstrated the feasibility of producing lipid from arabinose by genetic modification of the rhodococci strain.     

Engineering levoglucosan metabolic pathway in <Emphasis Type="Italic">Rhodococcus jostii</Emphasis> RHA1 for lipid production

Oleaginous strains of Rhodococcus including R. jostii RHA1 have attracted considerable attention due to their ability to accumulate triacylglycerols (TAGs), robust growth properties and genetic tractability. In this study, a novel metabolic pathway was introduced into R. jostii by heterogenous expression of the well-characterized gene, lgk encoding levoglucosan kinase from Lipomyces starkeyi YZ-215. This enables the recombinant R. jostii RHA1 to produce TAGs from the anhydrous sugar, levoglucosan, which can be generated efficiently as the major molecule from the pyrolysis of cellulose. The recombinant R. jostii RHA1 could grow on levoglucosan as the sole carbon source, and the consumption rate of levoglucosan was determined. Furthermore, expression of one more copy of lgk increased the enzymatic activity of LGK in the recombinant. However, the growth performance of the recombinant bearing two copies of lgk on levoglucosan was not improved. Although expression of lgk in the recombinants was not repressed by the glucose present in the media, glucose in the sugar mixture still affected consumption of levoglucosan. Under nitrogen limiting conditions, lipid produced from levoglucosan by the recombinant bearing lgk was up to 43.54 % of the cell dry weight, which was comparable to the content of lipid accumulated from glucose. This work demonstrated the technical feasibility of producing lipid from levoglucosan, an anhydrosugar derived from the pyrolysis of lignocellulosic materials, by the genetically modified rhodococci strains.     

Xylem anatomy and calculated hydraulic conductance of four <Emphasis Type="Italic">Nothofagus</Emphasis> species with contrasting distribution in South-Central Chile

Nothofagus obliqua, N. dombeyi, N. alpina and N. antarctica are characteristic tree species of the temperate forests on the western slopes of the Andes with centres of distribution that differ in their temperature and moisture regimes. We tested branch wood from co-occurring specimens of these species for the inherent differences in xylem anatomy and theoretical hydraulic conductance to evaluate their resistance to drought or frost. The hydraulic conductivity of the xylem was calculated using a modified Hagen–Poiseuille equation and related to wood density. Conduit dimensions were used to predict the water potential that would cause 50 % loss of hydraulic conductivity (Ψ ₅₀). Nothofagus alpina, which mainly grows at sites with low frost frequency, exhibited the largest conduits and the highest mean values for conduit area, fraction of conduit area in the cross-section and hydraulic conductivity, but the lowest wood density. Opposite relationships were found in the plastic N. antarctica, whose xylem seems to be least vulnerable to freezing-induced, but also to drought-induced embolism. Calculated Ψ ₅₀ was highest (least negative) in N. alpina, indicating a relatively high susceptibility to cavitation. The xylem of the thermophilic N. obliqua and of N. dombeyi, which mainly occurs under oceanic climate, but can also survive at sporadically dry and warm sites, is not particularly adapted to periods of drought stress. Across all species, wood density was negatively correlated with the calculated hydraulic conductance. The xylem traits of N. alpina might contribute to its relatively high growth rate and facilitate its spread into forest gaps.     

Metabolic reconstruction and experimental verification of glucose utilization in <Emphasis Type="Italic">Desulfurococcus amylolyticus</Emphasis> DSM 16532

Desulfurococcus amylolyticus DSM 16532 is an anaerobic and hyperthermophilic crenarchaeon known to grow on a variety of different carbon sources, including monosaccharides and polysaccharides. Furthermore, D. amylolyticus is one of the few archaea that are known to be able to grow on cellulose. Here, we present the metabolic reconstruction of D. amylolyticus’ central carbon metabolism. Based on the published genome, the metabolic reconstruction was completed by integrating complementary information available from the KEGG, BRENDA, UniProt, NCBI, and PFAM databases, as well as from available literature. The genomic analysis of D. amylolyticus revealed genes for both the classical and the archaeal version of the Embden-Meyerhof pathway. The metabolic reconstruction highlighted gaps in carbon dioxide-fixation pathways. No complete carbon dioxide-fixation pathway such as the reductive citrate cycle or the dicarboxylate-4-hydroxybutyrate cycle could be identified. However, the metabolic reconstruction indicated that D. amylolyticus harbors all genes necessary for glucose metabolization. Closed batch experimental verification of glucose utilization by D. amylolyticus was performed in chemically defined medium. The findings from in silico analyses and from growth experiments are discussed with respect to physiological features of hyperthermophilic organisms.     

Cultivation of the microalga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis> for biofuels production and other industrial applications (a review)

Microalgae cultivation for biofuels production and other applications has gained considerable interest recently. Despite their simple structures, microalgae can accumulate significant amounts of neutral lipids per dry cell weight compared to other energy crops. Neochloris oleoabundans is a promising microalga known for its high lipid content and biomass growth rate compared to other species cultivated for biofuels synthesis; therefore, it is considered as a suitable candidate for biodiesel synthesis. This review paper covers several key aspects associated with the cultivation and applications of the microalga N. oleoabundans. Biomass composition, factors affecting the growth, and biomass and lipid productivities of this species were addressed. In addition, different growth conditions as well as alternative readily available nutrient media to support the growth of N. oleoabundans were presented in this review.     

Enhancement of Pyruvate Productivity in <Emphasis Type="Italic">Candida glabrata</Emphasis> by Deleting the <Emphasis Type="Italic">CgADE13</Emphasis> Gene to Improve Acid Tolerance

Acid tolerance is one of the critical factors to evaluate the quality of the industrial production strains, especially organic acid producing microorganisms. To circumvent this problem, we investigated the physiological function of adenylosuccinate lyase in AMP metabolism from Candida glabrata by deleting the corresponding gene, CgADE13. At pH 4.0, CgADE13 deletion resulted in a 68.3% and 112.0% increase in biomass and cell viability compared to those of wild type strain (wt), respectively. In addition, CgADE13 deletion also protected cell morphology and counteracted ROS production. Further, the intracellular ATP level of strain Cgade13Δ was decreased by 25.0%, and its H+-ATPase activity was increased by 15.0%. Finally, pyruvate production with strain Cgade13Δ in a 30-L batch bioreactor at pH 4.0 reached 53.9 g/L, and pyruvate productivity was increased by 166.7% compared to that of wt. This is the first report regarding tolerance engineering of C. glabrata for enhancing pyruvate productivity, which provides a good starting point for metabolic engineering to achieve the industrial production of other chemicals.     

Heat and hypoxia give a global invader, <Emphasis Type="Italic">Gambusia holbrooki,</Emphasis> the edge over a threatened endemic fish on Australian floodplains

Deciphering the mechanisms by which climate change interacts with invasive species to affect biodiversity is a major challenge of global change biology. We conducted experiments to determine whether the global invader, Gambusia holbrooki, was more resistant to high water temperature (heat) and low dissolved oxygen (hypoxia) than a threatened native fish, Nannoperca australis. Metabolic experiments conducted at 25 and 29 °C showed that G. holbrooki had at least four times the capacity for metabolic depression during hypoxia than N. australis. An increase in environmental temperature from 25 to 29 °C had no significant impact on the critical oxygen tension, P _crit, of G. holbrooki, but significantly and strongly increased P _crit of N. australis. Gambusia holbrooki also had a lower Q ₁₀ of standard metabolic rate than N. australis. Our results indicate that G. holbrooki have physiological traits conferring greater resistance to hypoxia than N. australis, and as temperature increases, the resistance of N. australis to hypoxia was more eroded than that of G. holbrooki. Intensive monitoring of the temperature and dissolved oxygen dynamics of wetlands showed that contemporary heat waves are already causing conditions that might give G. holbrooki the edge over N. australis on Australian floodplains. Our study adds weight to recent anecdotal reports of drought and heat waves causing localised extinction of N. australis, but the proliferation of G. holbrooki.     

Engineering the growth pattern and cell morphology for enhanced PHB production by <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli JM109?envC?nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109?minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109?envC?nlpD and E. coli JM109?minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109?minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109?envC?nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.     

Comparison of optimal conditions for mass production of carboxymethylcellulase by <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 with other recombinants in pilot-scale bioreactor

The optimal conditions for mass production of carboxymethylcellulase (CMCase) by E. coli JM109/A-68 were investigated and compared with other E. coli JM109 recombinants producing CMCase. The optimal agitation speed and aeration rate for cell growth of E. coli JM109/A- 68 were 500 rpm and 0.50 vvm in a 7 L bioreactor, whereas those for production of CMCase were 416 rpm and 0.95 vvm. The optimal vessel pressures for cell growth as well as production of CMCase in a 100 L bioreactor were 0.04 MPa. The maximal production of CMCase by E. coli JM109/A-68 under the optimized conditions in a 100 L bioreactor was 11.0 times higher than its wild type, B. velezensis A-68. Optimal conditions for mass production of CMCase by recombinants were different from those for wild strains. The higher production of CMCase by E. coli JM109/A-68 and other recombinant of E. coli seemed to result from its higher cell growth under the optimal conditions for dissolved oxygen and its mixed-growth associated production pattern compared to the growthassociated production of B. velezensis A-68.     

Identification and functional characterization of <Emphasis Type="Italic">APRR2</Emphasis> controlling green immature fruit color in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Nitrogen (N) is a macronutrient essential for plant growth and development. Meanwhile, grafting is a method used to alleviate stress tolerance of various biotic and abiotic factors. This study aims to investigate how pumpkin grafting (PG) improves N use efficiency of watermelon. A commercial watermelon cultivar “Zaojia 8424” [Citrullus lanatus (Thunb.) Matsum. and Nakai.] was self-grafted and then grafted onto pumpkin (Cucurbita maxima?×C. moschata) rootstock cv. Qingyan Zhenmu No. 1. The grafted plants were exposed to two levels of N (9 and 0.2 mM) under hydroponic conditions. The grafted plants were harvested at days 11 and 22 after low N (0.2 mM) treatment. PG improved the N use efficiency of watermelon scion through the vigorous root system of pumpkin rootstock that enhanced the uptake and accumulation of N, P, K, Ca, Mg, B, and Mn in watermelon. Gene expressions of nitrate reductase (Cla002787, Cla002791, and Cla023145) and nitrite reductase (Cla013062) genes were increased, promoting N assimilation. Mesophyll thickness and SPAD index (relative chlorophyll measurement) were also improved. Furthermore, pumpkin rootstock also enhanced the supply of zeatine riboside (ZR) and isopentenyl adenosine (iPA) in the leaves, promoting shoot growth. All these lead to improved plant growth and nitrogen use efficiency of pumpkin rootstock-grafted watermelon plants.     

Modulation of proline metabolic gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under water-stressed conditions by a drought-mitigating <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ ¹ -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ ¹ -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.     

Plant growth promoting potential of bacterial endophytes in novel association with <Emphasis Type="Italic">Olea ferruginea</Emphasis> and <Emphasis Type="Italic">Withania coagulans</Emphasis>

Microbially unexplored medicinal plants can have a genetically diverse microbial population with multi-functional plant growth promoting traits. In this aspect, 75 endophytic bacterial isolates with plant growth promoting traits were isolated from Withania coagulans Dunal and Olea ferruginea Royal. Many of these bacteria were able to solubilize phosphate, produce indole-3-acetic acid, ammonia as well as hydrogen cyanide, synthesize extracellular enzymes and show antagonistic activities against plant pathogenic fungi under in vitro conditions. These isolates were also characterized by morphological and biochemical analysis. Furthermore, four representative isolates with pronounced plant growth promoting activities were identified as Enterobacter cloacae, Enterobacter dissolvens, Enterobacter hormaechei and Cronobacter sakazakii by 16S rDNA sequencing analysis. This work for the first time, reported the isolation of endophytic bacteria, the novel association form selected plants, Withania coagulans and Olea ferruginea. The explored endophytes might have great potential in the field of biocontrol and plant growth promoting for sustainable agricultural practices.     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Changes in growth and composition of the marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> in response to changing sodium bicarbonate concentrations

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L^?1 NaHCO₃ added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO₃ is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly (p?<?0.05) higher in the NaHCO₃-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO₃] during the GP. During the LP, cell densities were generally higher in the NaHCO₃-treated cells compared with controls. F _V/F _M (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO₃ and significantly different values between controls and 5.0 g L^?1 NaHCO₃ at the end of the GP (p?<?0.05). F _V/F _M was variable between treatments in P. tricornutum (0.3–0.65) but less so in N. salina for (0.5–0.7) regardless of [NaHCO₃]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2?±?6.5 and 4.4?±?2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L^?1 NaHCO₃ treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L^?1 NaHCO₃. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO₃]; there was three times more crude protein in the highest NaHCO₃ (5.0 g L^?1) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03?±?0.12 and 10.16?±?0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO₃] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO₃] (t tests; p?<?0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO₃ treatments. These findings reveal the importance of considering NaHCO₃ as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels.     

Physiological and Morphological Characteristics of Acidophilic Bacteria <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus thiooxidans</Emphasis>, Members of a Chemolithotrophic Microbial Consortium

A thermoacidophilic consortium of chemolithotrophic microorganisms oxidizing the concentrate of high-pyrrhotite pyrite?arsenopyrite ore at 38–40°C was isolated. The most active members of the consortium were identified as Leptospirillum ferriphilum, Acidithiobacillus thiooxidans, Ferroplasma acidiphilum, and Sulfobacillus thermotolerans. Leptospirillum and Thiobacillus species were the most numerous members of the consortium and had the highest activity of ferrous iron and sulfur oxidation, respectively. The optimal temperature values for the growth of both isolates were within 35–38°C. The optimal ranges of initial pH were 1.0–1.2 and 1.75–1.85 for leptospirilla and 1.7–3.3 for thiobacilli with the pH optimum of 1.9. Significant polymorphism and specific cyclic growth with formation of vibrios, spirilla, rods with different end shape, spiral filaments, numerous “pseudococci,” and densely packed spiral filaments surrounded by a sheath were revealed for the Leptospirillum isolate. Two latter morphoforms of L. ferriphilum were not previously described. Differences in ability of the morphoforms to oxidize Fe²⁺ were revealed. For the first time, the possibility of growth in the presence of organic substances was demonstrated for A. thiooxidans. The rates of growth and substrate oxidation, cell size, and the maximal cell yield decreased insignificantly in comparison with the lithoautotrophic strain.