H2-CO2 Recirculation and pH Control for Growth of Methanogens in Mass Culture

We modified a fermentor (10-liter liquid volume) for the growth of anaerobic, H₂-CO₂-catabolizing bacteria. Gas in the fermentor (ca. 10% CO₂, 50% H₂, 40% CH₄) was recirculated by a diaphragm pump. During growth, the gas composition was maintained by the addition of a mixture of 80% H₂ and 20% CO₂, and this addition was controlled by a pH auxostat. During gas addition, gas was discharged from the recirculating gas stream and was collected by the displacement of an acidified salt solution.     

Determination of the interparticular effective diffusion coefficient for CO(2) and O(2) in solid state fermentation

A simple experimental diffusion controlled fermentor (DCF), coupled with the use of a mathematical model based on mass balance, is proposed to measure the variation of the gas (CO(2) and O(2)) diffusion coefficients in solid state fermentation. The DCF was packed with an ion-exchange resin impregnated with a nutritive medium and inoculated with Aspergillus niger. The growth conditions in the DCF were very similar to those found in equipment operated with convective oxygen supply. The diffusion coefficient was shown to be very dependent on the biomass concentration within the solid state fermentor, and attained values of less than 5% of the molecular diffusion in air when the biomass in the fermentor reached 27 mg dry/g dry support.     

Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Chlorobium limicola forma thiosulfatophilum: Biocatalyst in the Production of Sulfur and Organic Carbon from a Gas Stream Containing H(2)S and CO(2)

Chlorobium limicola forma thiosulfatophilum (ATCC 17092) was grown in a 1-liter continuously stirred tank reactor (800-ml liquid volume) at pH 6.8, 30 degrees C, saturated light intensity, and a gas flow rate of 23.6 ml/min from a gas cylinder blend consisting of 3.9 mol% H(2)S, 9.2 mol% CO(2), 86.4 mol% N(2), and 0.5 mol% H(2). This is the first demonstration of photoautotrophic growth of a Chlorobium sp. on a continuous inorganic gas feed. A significant potential exists for applying this photoautotrophic process to desulfurization and CO(2) fixation of gases containing acidic components (H(2)S and CO(2)).     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H(2)-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H(2)-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 +/- 0.19 mol%), a phospholipid pattern of type II, MK-9(H(4)) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t(d) = 8 h), H(2) plus CO(2) (t(d) = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H(2), nickel was not necessary. The optimum growth temperature was 65 degrees C; no growth was observed below 40 degrees C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70 degrees C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K(m) was 88 mul of CO liter and V(max) was 20.2 mul of CO h mg of protein. The threshold value of S. thermoautotrophicus of 0.2 mul of CO liter was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 mumol min mg of protein. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Enhanced photo-H2 production of R. faecalis RLD-53 by separation of CO2 from reaction system

The effect of different gases, CO(2) concentration, and separation of CO(2) from reaction system on photo-fermentation H(2) production was investigated by batch culture in this study. Experimental results showed that different gases (Ar,N(2),CO(2), and air) as gas phase have obviously affected on photo-H(2) production and a high concentration of CO(2) can inhibit the growth and H(2) evolution of Rhodopseudomonas faecalis RLD-53. When CO(2) concentration at 5%, cell increased most rapidly the specific growth rate of 0.489 g/l/h and the specific growth rate fell to be 0.265 g/l/h when CO(2) concentration at 40%. However, the growth of RLD-53 at CO(2) concentration of 60-100% was almost completely inhibited. At CO(2) concentrations of 5% and 10%, the maximum H(2) yield was 2.54 and 2.59 mol-H(2)/mol acetate, respectively, and it was similar with the control (2.61 mol-H(2)/mol acetate). H(2) not produced when CO(2) concentration at 60-100%. In conclusion, separation of CO(2) from reaction system can stimulate H(2) production in the entire photo-H(2) production process and H(2) yield increased about 12.8-18.85% than the control.     

Impact of fermentation rate changes on potential hydrogen sulfide concentrations in wine

The correlation between alcoholic fermentation rate, measured as carbon dioxide (CO2) evolution, and the rate of hydrogen sulfide (H2S) formation during wine production was investigated. Both rates and the resulting concentration peaks in fermentor headspace H2S were directly impacted by yeast assimilable nitrogenous compounds in the grape juice. A series of model fermentations was conducted in temperature-controlled and stirred fermentors using a complex model juice with defined concentrations of ammonium ions and/or amino acids. The fermentation rate was measured indirectly by noting the weight loss of the fermentor; H2S was quantitatively trapped in realtime using a pre-calibrated H2S detection tube which was inserted into a fermentor gas relief port. Evolution rates for CO2 and H2S as well as the relative ratios between them were calculated. These fermentations confirmed that total sulfide formation was strongly yeast strain-dependent, and high concentrations of yeast assimilable nitrogen did not necessarily protect against elevated H2S formation. High initial concentrations of ammonium ions via addition of diammonium phosphate (DAP) caused a higher evolution of H2S when compared with a non-supplemented but nondeficient juice. It was observed that the excess availability of a certain yeast assimilable amino acid, arginine, could result in a more sustained CO2 production rate throughout the wine fermentation. The contribution of yeast assimilable amino acids from conventional commercial yeast foods to lowering of the H2S formation was marginal.     

Biosynthesis of Poly(3-Hydroxyalkanoic Acid) Copolymer from CO(inf2) in Pseudomonas acidophila through Introduction of the DNA Fragment Responsible for Chemolithoautotrophic Growth of Alcaligenes hydrogenophilus

Pseudomonas acidophila is a bacterial strain producing a poly(3-hydroxyalkanoic acid) (PHA) copolymer from low-molecular-weight organic compounds such as formate and acetate. The genes responsible for PHA production were cloned in cosmid pIK7 containing a 14.8-kb HindIII fragment of P. acidophila DNA. With the aim of developing a means of producing a PHA copolymer from CO(inf2), cosmid pIK7 was introduced into a polymer-negative mutant of the chemolithoautotrophic bacterium Alcaligenes eutrophus PHB(sup-)4. However, the recombinant strain produced a homopolymer of 3-hydroxybutyric acid (polyhydroxybutyric acid) from CO(inf2). Since it was thought that the composition of the accumulated polymer might depend not on the PHA biosynthetic genes but on the metabolism of the host strain, a recombinant plasmid, pFUS, containing the genes for chemolithoautotrophic growth of the hydrogen-oxidizing bacterium A. hydrogenophilus was introduced into P. acidophila by conjugation. The recombinant plasmid pFUS was stably maintained in P. acidophila in the absence of chemolithoautotrophic or antibiotic selection. This pFUS-harboring strain possessed the ability to grow under a gas mixture of H(inf2), O(inf2), and CO(inf2) in a mineral salts medium, and PHA copolymer accumulation was confirmed by nuclear magnetic resonance spectral analysis. A gas chromatogram obtained by gas chromatography-mass spectrometry showed the composition of the polymer to be 52.8% 3-hydroxybutyrate, 41.1% 3-hydroxyoctanoate, and 6.1% 3-hydroxydecanoate. This is the first report of the production of a PHA copolymer from CO(inf2) as sole carbon source.     

Hydrogenogenic CO conversion in a moderately thermophilic (55 degrees C) sulfate-fed gas lift reactor: competition for CO-derived H(2)

Thermophilic (55 degrees C) sulfate reduction in a gas lift reactor fed with CO gas as the sole electron donor was investigated. The reactor was inoculated with mesophilic granular sludge with a high activity of CO conversion to hydrogen and carbon dioxide at 55 degrees C. Strong competition for H(2) was observed between methanogens and sulfate reducers, while the homoacetogens present consumed only small amounts of H(2). The methanogens appeared to be more sensitive to pH and temperature shocks imposed to the reactor, but could not be completely eliminated. The fast growth rates of the methanogens (generation time of 4.5 h) enabled them to recover fast from shocks, and they rapidly consumed more than 90% of the CO-derived H(2). Nevertheless, steep increases in sulfide production in periods with low methane production suggests that once methanogenesis is eliminated, sulfate reduction with CO-rich gas as electron donor has great potential for thermophilic biodesulfurization.     

Carbon Dioxide Control of Lag Period and Growth of Streptococcus sanguis

A carbon dioxide requirement for growth of Streptococcus sanguis was readily demonstrated in a fermentor where the gas atmosphere could be controlled. Growth at a maximum rate occurred immediately in response to the appropriate CO(2) concentration; growth stopped when CO(2) was deleted. Washed inocula consisting of exponentially growing cells required a minimum of 2.4% CO(2), postexponential phase cells needed 1.2 to 1.8% CO(2) immediately and 2.4% CO(2) shortly thereafter, whereas stationary phase cells required three sequential increases in CO(2) from 0.3 to 1.8 to 2.4% within the first 90 min of growth. These CO(2) concentrations permitted each inoculum to initiate growth immediately at the same maximum rate. These results also showed that physiologically "old" cells had the same capacity for growth as "young" cells when the CO(2) concentrations were appropriate for the type of inoculum. Continued exponential growth of the culture at the same optimum rate required 2.4% CO(2). Lower concentrations of CO(2) were rate limiting and the resulting exponential rate was proportional to the CO(2) concentration. The "normal" lag period of S. sanguis appears to be an artifact induced by a CO(2) deficiency.     

Peptostreptococcus productus strain that grows rapidly with CO as the energy source

Anaerobic bacteria were enriched with a sewage digestor sludge inoculum and a mineral medium supplemented with B-vitamins and 0.05% yeast extract and with a 50% CO-30% N2-20% CO2 (2 atm [202 kPa]) gas phase. Microscopic observation revealed an abundance of gram-positive cocci, 1.0 by 1.4 micron, which occurred in pairs or chains. The coccus, strain U-1, was isolated by using roll tubes with CO as the energy source. Based on morphology, sugars fermented, fermentation products from glucose (H2, acetate, lactate, and succinate), and other features, strain U-1 was identified as Peptostreptococcus productus IIb (similar to the type strain). The doubling time with up to 50% CO was 1.5 h; acetate and CO2 were the major products. In addition, no significant change in the doubling time was observed with 90% CO. Some stock strains were also able to use CO, although not as well. Strain U-1 produced acetate during growth with H2-CO2. Other C1 compounds did not support growth. Most probable numbers of CO utilizers morphologically identical with strain U-1 were 7.5 X 10(6) and 1.1 X 10(5) cells per g for anaerobic digestor sludge and human feces, respectively.     

Peptostreptococcus productus strain that grows rapidly with CO as the energy source.

Anaerobic bacteria were enriched with a sewage digestor sludge inoculum and a mineral medium supplemented with B-vitamins and 0.05% yeast extract and with a 50% CO-30% N2-20% CO2 (2 atm [202 kPa]) gas phase. Microscopic observation revealed an abundance of gram-positive cocci, 1.0 by 1.4 micron, which occurred in pairs or chains. The coccus, strain U-1, was isolated by using roll tubes with CO as the energy source. Based on morphology, sugars fermented, fermentation products from glucose (H2, acetate, lactate, and succinate), and other features, strain U-1 was identified as Peptostreptococcus productus IIb (similar to the type strain). The doubling time with up to 50% CO was 1.5 h; acetate and CO2 were the major products. In addition, no significant change in the doubling time was observed with 90% CO. Some stock strains were also able to use CO, although not as well. Strain U-1 produced acetate during growth with H2-CO2. Other C1 compounds did not support growth. Most probable numbers of CO utilizers morphologically identical with strain U-1 were 7.5 X 10(6) and 1.1 X 10(5) cells per g for anaerobic digestor sludge and human feces, respectively.     

Growth of Rhodospirillum rubrum on synthesis gas: conversion of CO to H2 and poly-beta-hydroxyalkanoate

To examine the potential use of synthesis gas as a carbon and energy source in fermentation processes, Rhodospirillum rubrum was cultured on synthesis gas generated from discarded seed corn. The growth rates, growth and poly-beta-hydroxyalkanoates (PHA) yields, and CO oxidation/H(2) evolution rates were evaluated in comparison to the rates observed with an artificial synthesis gas mixture. Depending on the gas conditioning system used, synthesis gas either stimulated or inhibited CO-oxidation rates compared to the observations with the artificial synthesis gas mixture. Inhibitory and stimulatory compounds in synthesis gas could be removed by the addition of activated charcoal, char-tar, or char-ash filters (char, tar, and ash are gasification residues). In batch fermentations, approximately 1.4 mol CO was oxidized per day per g cell protein with the production of 0.75 mol H(2) and 340 mg PHA per day per g cell protein. The PHA produced from R. rubrum grown on synthesis gas was composed of 86% beta-hydroxybutyrate and 14% beta-hydroxyvalerate. Mass transfer of CO into the liquid phase was determined as the rate-limiting step in the fermentation.     

The relationship between hydrogen gas and butanol production by Clostridium saccharoperbutylacetonicum

Two simultaneous fermentations were performed at 26 degrees C with simultaneous inocula using Clostridium saccharoperbutylacetonicum. Fermentation 1 prevented the gas formed by the biomass from escaping the fermentor while 2 allowed the gas formed to escape. Fermentor 1 provided for the production of butanol, acetone, and ethanol, while when the H(2) formed was allowed to escape with fermentor 2, neither butanol nor acetone were produced. Ethanol was also formed in both fermentors and began along with the initial growth of biomass and continued until the fermentations were complete. Butanol and acetone production began after biomass growth had reached a maximum and began to subside. The butanol-acetone-ethanol millimolar yields and ratios were 38:1:14 respectively. The fermentor 2 results show that a yield of 2.1 L H(2), 93 or 370 mmol H(2)/mol glucose, was formed only during the growing stage of growth; neither butanol nor acetone were produced; ethanol was formed throughout the fermentation, reaching a yield of 15.2 mmolar. It appears that hydrogen gas is required for butanol production during the resting stage of growth.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures

High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.     

Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor

The microalga incorporated photobioreactor is a highly efficient biological system for converting CO2 into biomass. Using microalgal photobioreactor as CO2 mitigation system is a practical approach for elimination of waste gas from the CO2 emission. In this study, the marine microalga Chlorella sp. was cultured in a photobioreactor to assess biomass, lipid productivity and CO2 reduction. We also determined the effects of cell density and CO2 concentration on the growth of Chlorella sp. During an 8-day interval cultures in the semicontinuous cultivation, the specific growth rate and biomass of Chlorella sp. cultures in the conditions aerated 2-15% CO2 were 0.58-0.66 d(-1) and 0.76-0.87 gL(-1), respectively. At CO2 concentrations of 2%, 5%, 10% and 15%, the rate of CO2 reduction was 0.261, 0.316, 0.466 and 0.573 gh(-1), and efficiency of CO2 removal was 58%, 27%, 20% and 16%, respectively. The efficiency of CO2 removal was similar in the single photobioreactor and in the six-parallel photobioreactor. However, CO2 reduction, production of biomass, and production of lipid were six times greater in the six-parallel photobioreactor than those in the single photobioreactor. In conclusion, inhibition of microalgal growth cultured in the system with high CO2 (10-15%) aeration could be overcome via a high-density culture of microalgal inoculum that was adapted to 2% CO2. Moreover, biological reduction of CO2 in the established system could be parallely increased using the photobioreactor consisting of multiple units.     

Some comments on respiratory quotient (RQ) determination from the analysis of exit gas from a fermentor

Recently, the respiratory quotient (RQ) of microbes measured in situ in a fermentor by exit-gas analysis has been used successfully, for instance, in a fed-batch culture of baker's yeast as a criterion to control the feeding rate.(1-3) It is significant here to keep RQ values close to unity throughout; any deviations of RQ from unity give rise to deterioration of the cell growth yield.However easy it might be to keep RQ values around unity by controlling the feeding rate, the question of whether or not RQ values determined by gas analysis at the fermentor exit could generally represent those in vivo deserves attention. Indeed, for a fermentation carried out at an alkaline side, gas analysis would give RQ values that differ remarkably from true values because of the medium's "storage" of CO(2) released from microbes. The purpose of this communication is to make clear those factors that would affect true RQ values in the analysis of exit gas from a fermentor.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Effect of H(2)-CO(2) on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H(2)-CO(2) and mixtures of H(2)-CO(2) and acetate or methanol was examined. The growth yield of strain 227 on H(2)-CO(2) in complex medium was 8.4 mg/mmol of methane produced. Growth in defined medium was characteristically slower, and cell yields were proportionately lower. Labeling studies confirmed that CO(2) was rapidly reduced to CH(4) in the presence of H(2), and little acetate was used for methanogenesis until H(2) was exhausted. This resulted in a biphasic pattern of growth similar to that reported for strain 227 grown on methanol-acetate mixtures. Biphasic growth was not observed in cultures on mixtures of H(2)-CO(2) and methanol, and less methanol oxidation occurred in the presence of H(2). In M. mazei the aceticlastic reaction was also inhibited by the added H(2), but since the cultures did not immediately metabolize H(2), the duration of the inhibition was much longer.