Isolation and characterization of sulphur-oxidizing Thiomonas sp. and its potential application in biological deodorization

AIMS: To isolate and characterize a sulphur-oxidizing bacterial strain from activated sludge and to evaluate its potential application in biological deodorization. METHODS AND RESULTS: A dominant sulphur-oxidizing bacterial strain, designated as strain SS, was isolated from an enrichment culture using thiosulphate as a sole energy source and CO2 as a sole carbon source. The cells of this organism were aerobic, rod-shaped, Gram-negative and motile. Strain SS could grow autotrophically, heterotrophically as well as mixotrophically. Autotrophic growth was observed at pH values ranging from 2.3 to 9.0. Phylogenetic analyses revealed that strain SS belonged to Group 1 of the genus Thiomonas, closely related to Thiomonas perometabolis and Thiomonas intermedia. The thiosulphate oxidation rates of strain SS at different pH values were evaluated in terms of oxygen uptake using a Micro-Oxymax respirometer. The results showed that the maximum oxidation rate of 5.65 mg l(-1) h(-1) occurred at 56 h of growth and pH 6.0. Continuous H2S removal study demonstrated that strain SS could remove more than 99% of H2S when the inlet concentration was below 58.6 ppm. Further increase of the inlet concentration to 118 ppm gave rise to a decline in the removal efficiency to ca 90%. CONCLUSIONS: The strong acidification of the culture medium during the later period could result in the deterioration of the growth activity and the metabolism activity of strain SS. In practical application, the problems caused by the end-product inhibition and the acidification can be alleviated by periodical replacement of culture medium with fresh medium. Given the physiological flexibility and the ability to remove H2S rapidly and efficiently, strain SS could be a good 'deodorizing' candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that Thiomonas species has been reported for biological deodorization application.     

Isolation and characterization of new facultative alkaliphilic Bacillus flexus strains from maize processing waste water (nejayote)

Aims: This work describes the isolation and characterization of two new alkaliphilic micro‐organisms present in nejayote. Methods and Results: Samples of fresh industrial nejayote were plated on nejayote medium and incubated for 4 days at 37°C. Isolates were identified based on morphological and physiological characteristics, as well as 16S rDNA sequence analysis. Two gram‐positive strains, NJY2 and NJY4, able to hydrolyse starch, xylan, and gelatin were isolated from nejayote. Comparative sequence analysis of 16S rDNA and phylogenetic studies indicate that the micro‐organisms studied were closely related to members of the Bacillus flexus species. The strains were identified as facultative alkaliphilic salt tolerant bacteria. Isolate NJY2 produced cell associated phenolic acid esterases, able to release ferulic acid from nixtamalised corn bran and ethyl and methyl esters. Conclusions: The isolated strains of B. flexus NJY2 and NJY4 showed important physiological properties to produce high‐value molecules from agroindustrial by‐products. Significance and Impact of the Study: This is the first report about the isolation of alkaliphilic micro‐organisms from nejayote and the first report of phenolic acid esterases synthesised by alkaliphiles. The new alkaliphilic micro‐organisms have potential application in the treatment and transformation of tortilla industry residues.     

Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135

An xylanase producing alkaliphilic Micrococcus sp was isolated from an alkaline soda lake. Xylose and xylan induced enzyme production but no activity was detected when it was grown using other carbohydrate sources. The level of xylanase production was higher in the presence of xylose than in the presence of xylan. The enzyme was purified to homogeneity and its molecular weight was estimated to be 56 kD on SDS-PAGE. The optimum temperature and pH for xylanase activity were 55°C and 7.5–9.0, respectively. Sixty per cent of the maximum activity was displayed at pH 11. The enzyme was very stable in the pH range of 6.5–10 and up to a temperature of 40°C. Xylanase activity was inhibited by Cu²⁺ and Hg²⁺. Received 03 October 1997/ Accepted in revised form 03 February 1998     

Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses

AIM: We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening. METHODS AND RESULTS: We used 7% NaCl glucose-yeast extract-peptone-fish extract broth and agar media (pH 9.5) for pour-plating and enrichment culture for 16 cheese samples produced in six European countries. HALAB were present in 9 of the 16 samples at < 20 --> 10(7) CFU g(-1). In three mould-ripened soft cheeses, HALAB counts ranged from 10(6) to 10(7) CFU g(-1) and were one order (two samples) and six orders (one sample) of magnitude greater than that of nonhaloalkaliphilic, common LAB, as enumerated on lactobacilli MRS agar. The 16S rRNA gene sequences (500 bp) of 51 of the 55 isolates examined were identical or similar to that of Marinilactibacillus psychrotolerans or Alkalibacterium olivapovliticus and related species, all of which are HALAB. CONCLUSIONS: HALAB of possible marine origin were present in various soft, semi-hard and semi-soft cheeses and were highly predominant in some mould-ripened cheeses. Significance AND IMPACT OF THE STUDY: HALAB of possible marine origin are members of the microflora of various cheeses and, when dominant, may play a role in the ripening of cheeses. Microbial analysis of LAB flora in cheeses should take into consideration the presence of HALAB.     

Isolation and characterization of sulfur-utilizing denitrifiers from the sulfur-oxidizing denitrification process

Of 14 potential sulfur-oxidizing strains, Pseudomonas sp. B21 and Agrobacterium sp. B19 were considered as denitrifiers. Under aerobic conditions, with S⁰ as electron donor, maximum cell growth rates were 0.022 (B21) and 0.043 h^–1 (B19). Both grew optimally at pH 7.5 and 28 °C. When NO₃-N was increased from 10 to 200 mg l^–1 the efficiency of nitrate removal of each strain gradually decreased, from 60 to 40%. Addition of suitable organic compounds (C/N < 4.2) increased the nitrate removal efficiencies of both strains, indicating their mixotrophic characters.     

Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio

A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.     

Fatty acid, compatible solute and pigment composition of obligately chemolithoautotrophic alkaliphilic sulfur-oxidizing bacteria from soda lakes

Salt adaptation in chemolithotrophic alkaliphilic sulfur-oxidizing strains belonging to genera Thioalkalimicrobium and Thioalkalivibrio has been studied by determination of salt-dependent changes in fatty acid and compatible solute composition. In both alkaliphilic groups, represented by the low salt-tolerant Thioalkalimicrobium aerophilum strain AL 3T and the extremely salt-tolerant Thioalkalivibrio versutus strain ALJ 15, unsaturated fatty acids predominate over saturated fatty acids. In strain AL 3T, C18:1, C16:0 and C16:1 were the dominant fatty acids. In strain ALJ 15, the concentrations of C18:1 and C19cyclo were salt-regulated in an inverse proportional relationship, suggesting the stimulation of cyclopropyl-synthetase activity. Squalene has been found in substantial amounts only in strain ALJ 15. Ectoine and glycine betaine were found to be the main osmolytes in Thioalkalimicrobium aerophilum and Thioalkalivibrio versutus, respectively. The production of ectoine and glycine betaine was positively correlated with the salt concentration in the growth medium. A novel type of membrane-bound yellow pigments was uniformly detected in the extremely salt-tolerant strains of Thioalkalivibrio with a backbone consisting of C15-polyene, whose specific concentration correlated with increasing salinity of the growth medium. The results suggest that the mechanisms of haloalkaliphilic adaptation in Thioalkalimicrobium sp. and Thioalkalivibrio sp. involve the production of cyclopropane fatty acids, organic compatible solutes and, possibly specific pigments.     

Lipase-producing microorganisms from a Kenyan alkaline soda lake

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.     

Biofiltration of reduced sulphur compounds and community analysis of sulphur-oxidizing bacteria

The present work aims to use a two-stage biotrickling filters for simultaneous treatment of hydrogen sulphide (H₂S), methyl mercaptan (MM), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS). The first biofilter was inoculated with Acidithiobacillus thiooxidans (BAT) and the second one with Thiobacillus thioparus (BTT). For separate feeds of reduced sulphur compounds (RSC), the elimination capacity (EC) order was DMDS > DMS > MM. The EC values were 9.8 g_MM-S/m³/h (BTT; 78% removal efficiency (RE); empty bed residence time (EBRT) 58 s), 36 g_DMDS-S/m³/h (BTT; 94.4% RE; EBRT 76 s) and 57.5 g_H2S-S/m³/h (BAT; 92% RE; EBRT 59 s). For the simultaneous removal of RSC in BTT, an increase in the H₂S concentration from 23 to 293 ppmv (EBRT of 59 s) inhibited the RE of DMS (97-84% RE), DMDS (86-76% RE) and MM (83-67% RE). In the two-stage biofiltration, the RE did not decrease on increasing the H₂S concentration from 75 to 432 ppmv.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

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.     

Isolation and characterization of low hydrogen sulphide producing wine yeast

Variants of the wine yeast Saccharomyces cerevisiae var. montrachet no. 522 which retain more and excrete less hydrogen sulphide (H₂S) into the growth medium have been isolated and characterized. Wine produced using these low excretors has been found to contain less H₂S.     

Isolation of obligately alkaliphilic magnetotactic bacteria from extremely alkaline environments

Large numbers of magnetotactic bacteria were discovered in mud and water samples collected from a number of highly alkaline aquatic environments with pH values of ≈ 9.5. These bacteria were helical in morphology and biomineralized chains of bullet-shaped crystals of magnetite and were present in all the highly alkaline sites sampled. Three strains from different sites were isolated and cultured and grew optimally at pH 9.0-9.5 but not at 8.0 and below, demonstrating that these organisms truly require highly alkaline conditions and are not simply surviving/growing in neutral pH micro-niches in their natural habitats. All strains grew anaerobically through the reduction of sulfate as a terminal electron acceptor and phylogenetic analysis, based on 16S rRNA gene sequences, as well as some physiological features, showed that they could represent strains of Desulfonatronum thiodismutans, a known alkaliphilic bacterium that does not biomineralize magnetosomes. Our results show that some magnetotactic bacteria can be considered extremophilic and greatly extend the known ecology of magnetotactic bacteria and the conditions under which they can biomineralize magnetite. Moreover, our results show that this type of magnetotactic bacterium is common in highly alkaline environments. Our findings also greatly influence the interpretation of the presence of nanometer-sized magnetite crystals, so-called magnetofossils, in highly alkaline environments.     

Isolation and characterization of novel psychrophilic,neutrophilic, Fe-oxidizing,chemolithoautotrophic alpha- and gamma-proteobacteria from the deep sea

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS(2)), basalt glass ( approximately 10 wt% FeO), and siderite (FeCO(3)), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10 degrees C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are gamma-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are alpha-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.     

Isolation and characterization of autotrophic, hydrogen-utilizing, perchlorate-reducing bacteria

Recent studies have shown that perchlorate (ClO₄ ^–) can be degraded by some pure-culture and mixed-culture bacteria with the addition of hydrogen. This paper describes the isolation of two hydrogen-utilizing perchlorate-degrading bacteria capable of using inorganic carbon for growth. These autotrophic bacteria are within the genus Dechloromonas and are the first Dechloromonas species that are microaerophilic and incapable of growth at atmospheric oxygen concentrations. Dechloromonas sp. JDS5 and Dechloromonas sp. JDS6 are the first perchlorate-degrading autotrophs isolated from a perchlorate-contaminated site. Measured hydrogen thresholds were higher than for other environmentally significant, hydrogen-utilizing, anaerobic bacteria (e.g., halorespirers). The chlorite dismutase activity of these bacteria was greater for autotrophically grown cells than for cells grown heterotrophically on lactate. These bacteria used fumarate as an alternate electron acceptor, which is the first report of growth on an organic electron acceptor by perchlorate-reducing bacteria.     

Isolation of biosurfactant-producing bacteria,product characterization,and evaluation

A gram-positive, nonfermentative, rod-shaped bacterium designated ST-5, identified as Rhodococcus, was isolated from Kuwait soil. Grown on hydrocarbon, such as kerosene and n-paraffin, the bacterium produced surface-active compounds (biosurfactants). Measurements of surface tension, critical micelle dilution and emulsifying activity indicated that the biosurfactant is produced as a primary metabolite. The ST-5 culture surface-active component is mainly glycolipid in nature. Whole-culture broth dropped surface tension to values below 27 mN/m and was stable during exposure to high salinity (10% NaCl), elevated temperatures (120°C for 15 min) and a wide range of pH values. The culture broth was effective in recovering up to 86% of the residual oil from oil-saturated sand packs, indicating potential value in enhanced oil-recovery processes.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

Isolation and characterization of oil-desulphurizing bacteria

The objective of this study was to isolate local bacterial strains capable of removing sulphur from oil fractions without degrading the hydrocarbon. Oil biodesulphurization is an important step in combating pollution problems emanating from burning fossil fuels. Organisms which survive on oil are plentiful in local Kuwaiti soils; however, those that selectively only attack the carbon–sulphur bond are more difficult to find. Three strains were isolated based on their ability to use dibenzothiophene (DBT) as a sole source of sulphur for growth at 30 °C. Similar to other biodesulphurization organisms, the strains convert DBT to [2-hydroxybiphenyl (2-HBP) as detected by gas chromatography (GC). The specific desulphurization activity was in the range 5–13 mol 2-HBP/g-cell × h. Identification of the strains, based on 16 rRNA gene sequence similarity, showed the strains to be Rhodococcus erythropolis and Rhodococcus globerulus. The biodesulphurization activity was enhanced by promoting oxidore-ductase enzyme co-expression through the addition of a carbon source. The desulphurization was limited by the availability of DBT to the organism. Interfacial mass transfer through the aqueous-organic layer was confirmed to be a limiting factor.