Hyperthermophilic Anaerobic Nitrate-Dependent Fe(II) Oxidization by Tibetan Hot Spring Microbiota and the Formation of Fe Minerals

Fe(II) in geothermal fluids was among the most important electron and energy sources for extremophiles and early life, and microbial oxidation of Fe(II) in turn contributed to the global Fe deposits such as banded iron formation (BIF). However, information was rare on Fe(II) bio-oxidation and consequent mineral formation in geothermal systems. In the present study, we investigated the anaerobic nitrate-depending Fe(II) oxidation (ANDFO) in the Tibetan hot springs with temperature ranging 52–86°C. ANDFO cultivation was established by inoculating sediments from the studied hot springs. Positive ANDFO reaction was observed in the cultures from three high-temperature hot springs (>80°C). Phylogenetic analysis showed that bacteria in the three obtained ANDFO cultures were mainly affiliated with phyla of Betaproteobacteria, Alphaproteobacteria, and Firmicutes. In the obtained ANDFO cultures, ferrous iron oxidation occurred with nitrate reduction, accompanied with the formation of magnetite and/or siderite, which could be finished within one week. The resulting euhedral magnetite was at the micrometer scale, which was larger in size and showed better crystallinity than its counterparts (usually <1?µm) formed by chemical reactions. Thus, it can be concluded that ANDFO bacteria and denitrifiers played important roles in the magnetite and siderite precipitation in the studied Tibetan hot springs. The coupling between Fe(II) oxidation and nitrate reduction mediated by thermophiles might provide a new mechanism for euhedral magnetite and siderite deposition in BIFs during the Precambrian period.     

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms

H₂-producing microorganisms are a promising source of sustainable biohydrogen. However, most H₂-producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H₂ exist, common H₂ sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity (TC) sensors and developed a parallelized, online H₂ monitoring for time-efficient characterization of H₂ production by anaerobes. Since TC sensors are nonspecific for H₂, the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO₂ concentrations. The systems' measurement range was validated with two anaerobes: a high H₂-producer (Clostridium pasteurianum) and a low H₂-producer (Phocaeicola vulgatus). Online monitoring of H₂ production in shake flask cultivations was demonstrated, and H₂ transfer rates were derived. Combined with online CO₂ and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H₂ production with the model anaerobes was gained. The presented online H₂ monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana, while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.     

Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii

Single inorganic carbon source was used for production of chemicals and fuels via fermentation processes. Clostridium ljungdahlii, a strictly anaerobic autotrophic bacterium, was grown on synthesis gas to produce acetate and ethanol from gaseous substrates. C. ljungdahlii was grown on a various concentrations of carbon monoxide with synthesis gas total pressures of 0.8–1.8 atm with an interval of 0.2 atm. The cell and product yields were 0.015 g cell/g CO and 0.41 g acetate/g CO, respectively. Formation of acetate was steady and the production trend was about the same for all of the gases initial pressure and at constant cell density. The ethanol concentration was enhanced by the initial presence of hydrogen and carbon dioxide in the liquid phase. There was no substrate inhibition while C. ljungdahlii was grown in the batch fermentation, even at high system pressure of 1.6 and 1.8 atm. A desired product molar ratio of ethanol:acetate (5:1) was achieved with total gas pressure of 1.6 and 1.8 atm.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.     

On-line monitoring of pullulanase production during continuous culture of Clostridium thermosulfurogenes

Summary An automated turbidimetric immunoassay for pullulanase from Clostridium thermosulfurogenes was developed for on-line bioprocess monitoring. Measurements are based on the turbidity caused by specific aggregation between pullulanase molecules present in the cultivation medium and antibodies against these enzymes. The time for one assay cycle is 3.0 min. Pullulanase concentrations between 10 units (U)/l and 1000 U/l could be measured. Standard deviations of less than 2% were found. The analysis system could be successfully employed for on-line product monitoring during a 240-h cultivation of C. thermosulfurogenes. An average correlation factor of 0.975 was obtained for the pullulanase data from on-line turbidimetric and the off-line reference assay, demonstrating the accuracy of this on-line method. Offprint requests to: Th. Scheper     

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors

The development of high‐performance photobioreactors equipped with automatic systems for non‐invasive real‐time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light‐induced fluorescence rise and the dark relaxation of the flash‐induced fluorescence yield (Qa^? ? re‐oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas‐tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long‐term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress‐sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real‐time monitoring of algal photosynthesis in photobioreactors.     

A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.     

Tracing carbon monoxide uptake by Clostridium ljungdahlii during ethanol fermentation using 13C-enrichment technique

Conversion of synthesis gas (CO and H₂) to ethanol can be an alternative, promising technology to produce biofuels from renewable biomass. To distinguish microbial utilization of carbon source between fructose and synthesis gas CO and to evaluate biological production of ethanol from CO, we adopted the ¹³C-enrichment of the CO substrate and hypothesized that the residual increase in δ¹³C of the cell biomass would reflect the increased contribution of ¹³C-enriched CO. Addition of synthesis gas to live culture medium for ethanol fermentation by Clostridum ljungdahlii increased the microbial growth and ethanol production. Despite the high ¹³C-enrichment in CO (99 atom % ¹³C), however, microbial δ¹³C increased relatively small compared to the microbial growth. The uptake efficiency of CO estimated using the isotope mass balance equation was also very low: 0.0014 % for the low CO and 0.0016 % for the high CO treatment. Furthermore, the fast production of ethanol in the early stage indicated that the presence of sugar in fermentation medium would limit the utilization of CO as a carbon source by C. ljungdahlii.     

Fed-batch cultivation of methanobacterium formicicum and its fluorometric monitoring

Fed-batch cultivation of Methanobacterium formicicum Z-281 was performed under substrate limiting conditions. Specific rate of culture growth amounted to 0.041 h^?1, cell yield related to substrate was 0.6 mg proteine/mM formate. Significant increase of coenzyme F₄₂₀ content in cells depending on time of cultivation was observed. Increase of the fluorescence intensity of the medium was also observed with the accumulation of biomass. These parameters were suggested for tracking culture growth. The possibility of application of fluorescent factors for methanogenic population control in the anaerobic digester is discussed.     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Optimization of selected cultivation parameters for Cordyceps guangdongensis

Aims: To increase the fruit body production of Cordyceps guangdongensis, selected cultivation conditions, especially nutritional parameters were optimized. Methods and Results: Cordyceps guangdongensis was inoculated on potato dextrose agar slants with pH values from 4·5 to 9·0 and cultivated in artificial media with different carbon and nitrogen supplements. Primordium formation in C. guangdongensis was favoured by slightly acidic conditions. Fruit body yields and biological efficiency (BE) recorded were all highest in cultures of C. guangdongensis supplemented with sucrose and KNO₃ as carbon and nitrogen supplements, respectively. Highest fruit body yields and BE values were recorded with C : N ratio of 12 : 1. The optimal medium consisted of (g l^?1) 20·0 sucrose, 4·0 soya bean powder, 5·0 beef extract and 10·0 KNO₃. Cultivation experiments using this medium confirmed its reliability; 18·35% of BE was obtained, compared with a calculated maximum BE of 18·65% based on orthogonal test data. Conclusions: Cordyceps guangdongensis preferred sucrose and potassium nitrate as best carbon and nitrogen supplements. It produced satisfying yield of fruit body with optimized medium. Significance and Impact of the Study: Optimized artificial cultivation conditions could promote the yield of C. guangdongensis and decreased the cost of production.     

The role of iron nutrition in photosynthesis and nitrogen assimilation in SCENEDESMUS QUADRICAUDA (Chlorophyceae) 1

The rate of photosynthesis and nitrate uptake are related to the iron concentration in the medium for the green alga Scenedesmus quadricauda (Turp.) Breb. Increased iron leads to changes in chlorophyll a concentration, carbon fixation rate per chlorophyll a and in vivo fluorescence characteristics. These parameters all indicate that the efficiency of photosynthesis is related to iron nutrition. Nitrate uptake rate is also a function of both Fe and light Iron-limited cultures had decreased nitrate uptake at low light whereas ammonium uptake was relatively constant. Iron-limited cultures fixed about twice as much carbon into protein relative to the total carbon fixed. Iron plays a crucial role in the bioenergetics of carbon and nitrogen metabolism and may be important in controlling patterns of productivity.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Enrichment of acetate-, propionate-, and butyrate-degrading co-cultures from the biofilm of an anaerobic fluidized bed reactor

Summary Scanning electron microphotographs from the biofilm of a pilot scale anaerobic fluid-ized-bed reactor fed with acetate, propionate, and butyrate as carbon sources showed a predominance of filamentous organisms resembling Methanothrix sp. which could be isolated as an al-most pure culture as well as a Methanosarcina strain. Three syntrophic cultures, enriched in the medium of Boone and Xun, contained four or five microscopically distinguishable microorganisms, among them Methanospirillum sp., Methanothrix sp., Methanosarcina sp., and rods of acetogenic bacteria degrading propionate or butyrate effectively.     

Engineering Clostridium ljungdahlii as the gas-fermenting cell factory for the production of biofuels and biochemicals

Clostridium ljungdahlii is a representative autotrophic gas-fermenting acetogen capable of converting CO₂ and CO into biomass and multiple metabolites. The carbon fixation and conversion based on C. ljungdahlii have great potential for the sustainable production of bulk biochemicals and biofuels using industrial syngas and waste gases. With substantial recent advances in genetic manipulation tools, it has become possible to study and improve the metabolic capability of C. ljungdahlii in gas fermentation. The product scope of C. ljungdahlii has been expanded through the introduction of heterologous production pathways followed by the modification of native metabolic networks. In addition, progress has been made in understanding the physiological and metabolic mechanisms of this anaerobe, contributing to strain designs for expected phenotypes. In this review, we highlight the latest research progresses regarding C. ljungdahlii and discuss the next steps to comprehensively understand and engineer this bacterium for an improved bacterial gas bioconversion platform.     

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.