Growth of the syntrophic anaerobic acetogen, strain PA-1, with glucose or succinate as energy source

Strain PA-1 (S. Barik, W.J. Brulla, and M.P. Bryant, Appl. Environ. Microbiol. 50:304-310, 1985) is an anaerobic, gram-negative rod that in pure culture decarboxylates succinate to propionate and that grows syntrophically as an acetogen with the H2 utilizer Methanospirillum hungatei if glucose, pyruvate, aspartate, or fumarate is provided. In pure culture, strain PA-1 grows optimally in a medium containing 5% ruminal fluid, 0.1% yeast extract, a 4:1 N2-CO2 gas phase, and 20 mM succinate. With the PA-1 plus M. hungatei coculture, good growth was obtained with 7.5 mM glucose and tryptophan could replace the yeast extract. Strain PA-1 in pure culture grew quite well in glucose medium if the large headspace was flushed intermittently with N2. Flushing with H2 inhibited this growth.     

PA-1, a Versatile Anaerobe Obtained in Pure Culture, Catabolizes Benzenoids and Other Compounds in Syntrophy with Hydrogenotrophs, and P-2 plus Wolinella sp. Degrades Benzenoids

Methanogenic enrichments catabolizing 13 mM phenylacetate or 4 mM phenol were established at 37°C, using a 10% inoculum from a municipal anaerobic digester. By using agar roll tubes of the basal medium plus 0.1% yeast extract-25 mM fumarate, a hydrogenotrophic lawn of Wolinella succinogenes and phenol or phenylacetate, strains P-2 and PA-1, respectively, were isolated in coculture with W. succinogenes. With the lawn deleted, PA-1 was isolated in pure culture. Strain P-2 is apparently a new species of anaerobic, motile, gram-negative, spindle-shaped, small rod that as yet has been grown only in coculture with W. succinogenes. It used phenol, hydrocinnamate, benzoate, and phenylacetate as energy sources. Product recovery by the coculture, per mole of phenol and 4.4 mol of fumarate used, included 2.03, 0.12, 0.08, and 3.23 mol, respectively, of acetate, propionate, butyrate, and succinate. Carbon recovery was 75% and H recovery was 80%, although CO₂ and a few other possible products were not determined. That P-2 is an obligate proton-reducing acetogen and possible pathways for its degradation of phenol are discussed. Strain PA-1 is apparently a new species of anaerobic, motile, relatively small, gram-negative rod. It utilized compounds such as phenylacetate, hydrocinnamate, benzoate, phenol, resorcinol, gallate, 4-aminophenol, 2-aminobenzoate, pyruvate, Casamino Acids, and aspartate as energy sources in coculture with W. succinogenes. Per mole of phenylacetate and 1.44 mol of fumarate used, 1.04, 0.53, and 0.78 mol of acetate, propionate, and succinate, respectively, were recovered from the coculture. Only about 50% of the carbon and H were recovered. In coculture with Methanospirillum hungatei, 0.96 mol of acetate and 0.25 mol of methane were recovered per mol of pyruvate used; 0.90 mol of acetate and 0.33 mol of methane, per mol of fumarate used; 0.93 mol of acetate and 0.54 mol of methane, per mol of aspartate used; and 1.71 mol of acetate and 0.57 mol of methane, per mol of glucose used. Carbon and H recoveries, assuming CO₂ and ammonia were produced in stoichiometric amounts, were 97 and 98% for pyruvate, 72.5 and 82% for fumarate, 96.5 and 98% for aspartate, and 61.8 and 76% for glucose. No explanation such as contamination could be found for the fact that the coculture PA-1 plus Wolinella sp. did not use glucose; after growth with M. hungatei on pyruvate, however, the latter coculture used glucose. The PA-1 pure culture produced 0.86 mol of propionate per mol of succinate used during growth. PA-1 produced a small amount of H₂. Strain PA-1 is the most versatile anaerobic bacterium yet known that catabolizes monobenzenoids in the absence of electron acceptors such as sulfate or nitrate.     

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 10(8) cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22-28 h at 28 degrees C. Cells were small, 0.5 x 5 microm in size, Gram-positive, and formed terminal oval spores. At 20 degrees C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO(2). At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture.     

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.     

Acetic acid and hydrogen metabolism during coculture of an acetic acid producing bacterium with methanogenic bacteria

Two microorganisms originally existing as a mixed culture obtained from an anaerobic digester fluid were separated for pure and coculture studies. One of these was motile, Gram-negative, and non-sporeforming, and it required yeast extract for growth and acetic acid production. This isolate produced H2 and did not need H2 and (or) CO2 for growth and acetate formation. The other isolate was a methanogen whick resembled Methanobacterium arbophilicum in morphology and substrate specificity. Coculture growth of the two isolates in yeast extract broth (80% N2--20% CO2 gas phase) indicated that the non-methanogen produced up to four to five times more H2 than when grown separately. Although the growth of the non-methanogen was not enhanced by the removal of H2 by the methanogen, the hydrogen produced was essential for the growth of methanogen. Similar results were obtained when the non-methanogen was cocultured with Methanospirillum hungatti GP1. Cultivation of the non-methanogen in the presence of M. hungatti GP1 (under abundance of 80% H2--20% CO2) indicated that the acetate produced was consumed by M. hungatii, without inhibiting the growth of the other culture.     

Impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum D-E7 was studied to determine the impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH in broth media (initial pH c. 5). Cultures with 0.1% nutritional supplement (tryptone, yeast extract, or soytone) yielded minimal growth, 2-3 mM oxalate, and a final culture pH of 4.2-4.8. In contrast, cultures with 0.1% nutritional supplement and 25 mM glucose yielded significant growth, minimal oxalate (<1 mM), and a final culture pH of 2.8-3.7. Similar trends were observed when glucose in 0.1% soytone cultures was replaced with 25 mM d-mannose, l-arabinose, or d-xylose. With 1% soytone-25 mM glucose cultures, growth and oxalate accumulation ( approximately 21 mM) occurred with little change in initial pH. This was not the case with 1% soytone-250 mM glucose cultures; increased glucose levels resulted in a decrease in oxalate accumulation ( approximately 7 mM) and in final culture pH (3.4). Time-course studies with these cultures revealed that oxalate accumulation was suppressed during growth when the culture pH dropped to <4. Overall, these results indicate that (1) the decrease in external pH (i.e. acidification) was independent of oxalate accumulation and (2) acidification coupled to glucose-dependent growth regulated oxalate accumulation by Sclerotinia sclerotiorum.     

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Characteristics of a New Strain of Bacillus popilliae Sporogenic In Vitro

Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.     

The effect of succinate on respiration, transamination, and pyruvate formation in cells of the yeast Dipodascus magnusii

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.     

Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures

The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.     

Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula

Veillonella parvula strain 259 (=DSM 2007) was able to grow on a mineral salts medium supplemented with (per litre) 1 g yeast extract, 1 g Tween-80, and 3 mg putrescine. 2 HCl, with 6 mM thioglycolate as reductant and lactate as growth substrate. Succinate did not serve as a growth substrate, but when added in conjunction with lactate, it was decarboxylated to propionate and resulted in a measurable increase in growth yield, corresponding to the formation of 2.4 g cell dry mass per mol succinate. A growth yield increase linked to succinate metabolism occurred only while lactate was also being metabolised. Experiments with cell suspensions showed that succinate decarboxylating activity was constitutive. Addition of succinate produced clear increases in cellular ATP levels in ATP-depleted washed cells.     

Oxidation of Elemental Sulfur by Sulfolobus acidocaldarius

Oxidation of elemental sulfur by Sulfolobus acidocaldarius, an autotroph which grows at high temperatures and low pH, was examined by use of (35)S-labeled elemental sulfur. When cultured at pH 3.2 and 70 C, S. acidocaldarius oxidized elemental sulfur essentially quantitatively to sulfuric acid. Oxidation rate paralleled growth rate and decrease in pH of the culture medium. Elemental sulfur was not oxidized under these conditions if the culture was poisoned with formaldehyde. During the growth phase, the proportion of cells attached to the sulfur crystals increased progressively, and in the later phases of growth over 10 times more cells were attached to sulfur than were free. Doubling times for eight strains growing on elemental sulfur varied from 37 to 55 h. The organism grows much more rapidly on yeast extract than on sulfur. In a medium containing both sulfur and yeast extract, sulfur oxidation was partially inhibited, although growth was excellent.     

Influence of Growth Conditions on the Production of a Bacteriocin, Pediocin AcH, by Pediococcus acidilactici H

The influence of growth parameters on the production of pediocin AcH by Pediococcus acidilactici H was studied. This strain produced large quantities of pediocin AcH in TGE broth (Trypticase [1%], glucose [1%], yeast extract [1%], Tween 80 [0.2%], Mn²⁺ [0.033 mM], Mg²⁺ [0.02 mM] [pH 6.5]) within 16 to 18 h at 30 to 37°C (final pH, 3.6 to 3.7). Pediocin AcH production was negligible when the pH of the medium was maintained at 5.0 or above, even in the presence of high cell mass.     

The Effect of Succinate on Respiration,Transamination, and Pyruvate Formation in Cells of the Yeast <Emphasis Type="Italic">Dipodascus magnusii</Emphasis>

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain

Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1. Received: November 18, 1999 / Accepted: December 2, 1999     

Hyphomicrobium as a component of the aquatic microflora--morphological and physiological studies on two strains

Aspects of the morphology, metabolism and physiology of two oligocarbophilic strains of Hyphomicrobium (H4K and S5K), isolated from the Plusssee, were studied. Both strains are able to grow on mineral salts media without added organic carbon sources. Strain H4K grows well even in double distilled water. The two strains cannot grow on mineral media in the absence of atmospheric CO2. No growth occurred also in air purified of organic carbon, in spite of the presence of CO2. On the contrary, there is good growth in the presence of some organic compounds and without atmospheric CO2, i.e., heterotrophic metabolism without CO2 assimilation is possible. Growth was enhanced in a methanol atmosphere, and by the addition of yeast extract, methylamine, peptone and glucose. In nutrient solutions containing acetate or formate as carbon source, growth of H4K begins only after an adaptation period of ca. 4 weeks.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.