Sulfidogenesis in Low pH (3.8–4.2) Media by a Mixed Population of Acidophilic Bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8–4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8–4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine

The biodiversity of culturable acidophilic microbes in three acidic (pH 2.7–3.7), metal-rich waters at an abandoned subarctic copper mine in central Norway was assessed. Acidophilic bacteria were isolated by plating on selective solid media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences. The dominant iron-oxidizing acidophile in all three waters was an Acidithiobacillus ferrooxidans -like eubacterium, which shared 98% 16S rDNA identity with the type strain. A strain of Leptospirillum ferrooxidans was obtained from one of the waters after enrichment in pyrite medium, but this iron oxidizer was below detectable levels in the acidic waters themselves. In two sites, there were up to six distinct heterotrophic acidophiles, present at 10³ ml⁻¹. These included Acidiphilium -like isolates (one closely related to Acidiphilium rubrum , a second to Acidiphilium cryptum and a third apparently novel isolate), an Acidocella -like isolate (96% 16S rDNA identity to Acidocella facilis ) and a bacterium that shared 94.5% 16S rDNA identity to Acidisphaera rubrifaciens. The other numerically significant heterotrophic isolate was not apparently related to any known acidophile, with the closest match (96% 16S rDNA sequence identity) to an acetogen, Frateuria aurantia . The results indicated that the biodiversity of acidophilic bacteria, especially heterotrophs, in acidic mine waters may be much greater than previously recognized.     

Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria.

Nineteen characterized strains and isolates of acidophilic heterotrophic bacteria were screened for their abilities to catalyse the reductive dissolution of the ferric iron mineral schwertmannite, under oxygen-limiting conditions. Acidocella facilis, Acidobacterium capsulatum, and all of the Acidiphilium, Acidocella and Acidobacterium-like isolates that grew in liquid cultures were able to reduce iron. In contrast, neither Acidisphaera rubrifaciens nor three Acidisphaera-like isolates tested were found to have the capacity for dissimilatory iron reduction. One of two iron-oxidizing Frateuria-like isolates also reduced iron under oxygen-limiting conditions. Microbial dissolution of schwertmannite was paralleled with increased concentrations of soluble ferrous iron and sulfate in microbial cultures, together with increased pH values and decreased redox potentials. While dissimilatory ferric iron reduction has been described previously for Acidiphilium spp., this is this first report of this capacity in Acidocella and the moderate acidophile Acidobacterium. The finding has significant implications for understanding of the biogeochemistry of acidic environments.     

Microbial recovery of vanadium by the acidophilic bacterium, <Emphasis Type="Italic">Acidocella aromatica</Emphasis>

Objective

To investigate the bioreduction of toxic pentavalent vanadium [vanadate; V(V)] in the acidophilic, Fe(III)-reducing obligately heterotrophic bacterium, Acidocella aromatica PFBC.

Results

Although the initial lag-phase of growth became extended with increasing initial V(V) concentrations, the final cell density during aerobic growth of A. aromatica PFBC was unaffected by up to 2 mM V(V). While strain PFBC is an aerobe, growth-decoupled PFBC cell suspensions directly reduced V(V) using fructose, both micro-aerobically and anaerobically, under highly acidic (pH 2) and moderately acidic (pH 4.5) conditions. Bio reduced V(IV) was subsequently precipitated even under micro-aerobic conditions, mostly by encrusting the cell surface. An anaerobic condition at pH 4.5 was optimal for forming and maintaining stable V(IV)-precipitates. Recovery of approx. 70 % of V(V) from the solution phase was made possible with V(V) at 1 mM.

Conclusions

The first case of direct V(V) reducing ability and its subsequent V recovery from the solution phase was shown in acidophilic prokaryotes. Possible utilities of V(V) bioreduction in acidic conditions, are discussed.

     

Biogeochemical cycling of iron and sulphur in leaching environments

Abstract: Bacterial dissimilatory reduction of iron and sulphur in extremely acidic environments is described. Evidence for reduction at two disused mine sites is presented, within stratified 'acid streamers' growths and in sediments from an acid mine drainage stream. A high proportion (approx. 40%) of mesophilic heterotrophic acidophiles were found to be capable of reducing ferric iron (soluble and insoluble forms) under microaerophilic and anoxic conditions. Mixed cultures of Thiobacillus ferrooxidans and Acidiphilium -like isolate SJH displayed cycling of iron in shake flask and fermenter cultures. Oxido-reduction of iron in mixed cultures was determined by oxygen concentration and availability of organic substrates. Some moderately thermophilic iron-oxidis- ing bacteria were also shown to be capable of reducing ferric iron under conditions of limiting oxygen when grown in glycerol/yeast extract or elemental sulphur media. Cycling of iron was observed in pure cultures of these acidophiles. Sulphate-reducing bacteria isolated from acid streamers could be grown in acidified glycerol/yeast extract media (as low as pH 2.9), but not in media used conventionally for their laboratory culture. An endospore-forming, non-motile rod resembling Desulfotomaculum has been isolated. This bacterium has a wide pH spectrum, and appears to be acid-tolerant rather than acidophilic.     

Pseudomonas aeruginosa (GRC1) as a strong antagonist of Macrophomina phaseolina and Fusarium oxysporum

A plant growth promotory bacterial strain, isolated from the potato rhizosphere, was characterized as Pseudomonas aeruginosa (GRC1). The isolate produced an hydroxamate type of siderophore after 48 h of incubation on tryptic soy medium under iron deficient conditions. The in vitro antifungal activity of P. aeruginosa was tested against two soil-borne plant pathogens, Macrophomina phaseolina and Fusarium oxysporum. The antagonistic behaviour of the isolate was tested by dual culture technique. The growth inhibition of M. phaseolina and F. oxysporum was 74.1% and 70.5%, respectively, after 5 days of incubation. The production of hydrocyanic acid and indole acetic acid was also recorded under normal growth conditions.     

An Acidophilic and a Neutrophilic Nitrobacter Strain Isolated from the Numerically Predominant Nitrite-Oxidizing Population of an Acid Forest Soil

Two physiologically and serologically distinct strains of chemoautotrophic nitrite-oxidizing bacteria were isolated as numerically predominant members of the nitrite-oxidizer population of an undisturbed forest soil with a pH range of 4.3 to 5.2. One isolate responded as a neutrophile, characteristic of the family Nitrobacteraceae, and cross-reacted strongly with fluorescent antibody to Nitrobacter strain Engel. The second isolate responded as an acidophile in pure culture, demonstrated maximal nitrite oxidation activity at pH 5.5, and had a pH tolerance range of pH 4.1 to 7.2. Nitrite oxidase in whole cells of the acidophile sustained activity to at least pH 3.5. Cell morphology of both strains typified the genus Nitrobacter in all respects when cultured at pH 7. However, under more acidic conditions the acidophile tended to elongate and at times appeared to branch. These data provide the first evidence for the existence of an acidophilic chemoautotrophic nitrifying bacterium. Isolation of the neutrophilic Nitrobacter strain reported here complements the earlier isolation of a neutrophilic Nitrosospira strain to provide further evidence of a prominent acid-intolerant population of chemoautotrophic nitrifiers in this acid forest soil.     

INFLUENCE OF OXIDATION-REDUCTION REDUCTANTS ON SALMONELLA TYPHIMURIUM GROWTH RATES AFTER INITIAL pH ADJUSTMENT AND ZINC COMPOUND ADDITION

The objective of this study was to examine the separate influence of acidic pH, reductants (cysteine and sulfide) and zinc compounds (Zn acetate and Zn sulfate) during anaerobic growth of a S. typhimurium poultry isolate in rich or minimal media. The anaerobic growth of a S. typhimurium poultry isolate in TSB medium was significantly inhibited by either acidic initial pH or higher concentrations of Zn acetate. S. typhimurium anaerobic growth in M9 minimal medium was significantly inhibited by either acidic pH or higher concentrations of Zn acetate or Zn sulfate. Most anaerobic growth rates of the S. typhimurium poultry isolate in acidic media and with higher concentrations of Zn acetate or Zn sulfate were less than 0.10 h^-1. The overall anaerobic growth rates of S. typhimurium were inhibited more in the presence (0.089 h^-1) of reductants than in the absence (0.102 h^-1) of reductants in M9 medium. Results in this study suggest that either increasing Zn concentration or decreasing initial pH can reduce growth rate of foodborne Salmonella under anaerobic growth conditions.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Isolation and characterization of Bacillus thioparus sp. nov., chemolithoautotrophic, thiosulfate-oxidizing bacterium

A novel bacterium, strain BMP-1(T), was isolated from a continuous wastewater treatment culture system operating with a bacterial consortium. Cells of the isolate were Gram-variable, aerobic, moderately halotolerant, motile and endospore-forming rods. Strain BMP-1(T) grew chemolithoautotrophically by oxidation of thiosulfate to sulfate with a growth yield of 1.07 g protein mol(-1) of thiosulfate consumed. DNA G+C content was 43.8 mol%. Its cell wall had peptidoglycan based on m-diaminopimelic acid, and the major component of fatty acid was C(15 : 0). The 16S rRNA gene analysis showed that strain belongs to the genus Bacillus, sharing a 99.5% of sequence similarity with Bacillus jeotgali CCM 7133(T). DNA-DNA hybridization between the isolate of this study and this strain was 44%. Thus, the inclusion of strain BMP-1(T) in the genus Bacillus is suggested as a novel species and the name Bacillus thioparus sp. nov. (Type strain BMP-1(T)=BM-B-436(T)=CECT 7196(T)) is proposed. The sequence of the 16S rRNA gene has been deposited in GenBank with accession number DQ371431.     

Ensuring constant oxygen supply during inoculation is essential to obtain reproducible results with obligatory aerobic acetic acid bacteria in vinegar production

Since obligatory aerobic acetic acid bacteria in vinegar production suffer even from short oxygen depletion during traditional precultivation steps, the reproducibility of results in the main culture is insufficient. Thus, the aim of this paper is to establish a reproducible small scale cultivation method for obligatory aerobic acetic acid bacteria at industrially relevant high ethanol and acetic acid concentrations by ensuring constant oxygen transfer in the whole inoculation procedure. An acetic acid bacteria preculture was drained off from a laboratory-scale bioreactor into an aerated mobile bubble column and then transferred to an already shaking RAMOS shake flask device. Whereas the respiration curves of the traditionally processed acetic acid bacteria cultures were low and greatly diverged, those of the preculture transferred first into the bubble column and then into the already shaking flasks were high and coincided with one another. Furthermore, shutting off aeration in the mobile bubble column led to a rapid decrease in bacterial activity. In conclusion, traditional precultivation steps are not suitable for obligatory aerobic acetic acid bacteria in vinegar production. Maintaining constant oxygen transfer is necessary to guarantee the reproducibility of main culture experiments with such bacteria.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Effect of sulphidogenesis on acid-phase digestion of waste activated sludge

This paper reports the influence of sulfate-reducing bacterial activity on acidogenic digestion of waste activated sludge (WAS). A series of experiments was conducted by feeding WAS to a 10-l, completely mixed, mesophilic reactor maintained at pH 5.0 under sulfidogenic and non-sulfidogenic conditions. Analyses of volatile fatty acids indicated that the productions of acetic and propionic acids were slightly increased by sulfidogenic activity at 218 mg/l level of sulfate when COD sulfate ratio was 55:1. Higher amounts of methanol, ethanol and hydrogen were observed in the non-sulfidogenic condition, but ammonia was lower than in the moderate sulfidogenic runs. At 0.63 kg VS/m³ d loading rate the hydrolysis was above 90% in both moderate sulfidogenic and non- sulfidogenic runs. The results of these experiments showed the possible influence of moderate sulfidogenesis in the protein degradation of WAS anaerobic digestion. However, the acid-phase digestion was adversely affected by increasing the sulfate concentration to 400 mg/l.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways

A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150–250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9–10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.     

Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding

Glycerol at 10–20 g l^–1 increased clavulanic acid production by Streptomyces clavuligerus in shake-flask culture. The biosynthesis of clavulanic acid continued for longer by feeding glycerol and production increased to 250 mg l^–1 compared with 115 mg l^–1 without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased further to about 280 mg l^–1 but also remained stable up to 130 h.     

Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source

Succinic acid was produced by fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. When cells were anaerobically cultured in a medium containing 6.5 g/L glycerol, a high succinic acid yield (133%) was obtained while avoiding the formation of by-product acetic acid. The gram ratio of succinic acid to acetic acid was 25.8:1, which is 6.5 times higher than that obtained using glucose (ca. 4:1) as a carbon source. Therefore, succinic acid can be produced with much less by-product formation by using glycerol as a carbon source, which will facilitate its purification. When glucose and glycerol were cofermented with the increasing ratio of glucose to glycerol, the gram ratio of succinic acid to acetic acid and succinic acid yield decreased, suggesting that glucose enhanced acetic acid formation irrespective of the presence of glycerol. Glycerol consumption by A. succiniciproducens required unidentified nutritional components present in yeast extract. By intermittently feeding yeast extract along with glycerol, a high succinic acid yield (160%) could be obtained while still avoiding acetic acid formation. This resulted in the highest ratio of succinic acid to acetic acid (31.7:1).     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。