Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.     

The removal of pyritic sulphur from coal by Leptospirillum-like bacteria

Summary The microbial oxidation of pyritic sulphur was studied in a 4.5-l airlift fermentor at pH 1.5 and 100 g/l pulp density. By microbial leaching with Leptospirillum-like bacteria 85% of the pyritic sulphur was removed within 40 days; 30% of the removed pyrite was oxidized to elemental sulphur, the rest being transformed to soluble sulphate. Accumulation of elemental sulphur could be avoided by using a mixed culture of Leptospirillum-like bacteria and Thiobacillus ferrooxidans. Apart from oxidation of elemental sulphur neither the pure nor the mixed culture showed a significant difference as to removal of pyrite.     

Evaluation of FT-IR spectroscopy as a tool to quantify bacteria in binary mixed cultures

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.     

Degradation of selected (bio-)surfactants by bacterial cultures monitored by calorimetric methods

The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.     

Metabolite production during the biodegradation of the surfactant sodium dodecyltriethoxy sulphate under mixed-culture die-away conditions

Sodium dodecyltriethoxy sulphate (SDTES), either pure or as a component of commercial surfactant mixtures, underwent rapid primary biodegradation by mixed bacterial cultures in OECD screen and river-water die-away tests. Inoculation of [35S]SDTES-containing solutions with OECD screen test media acclimatized to surfactants or their degradation products led to production of various 35S-labelled glycol sulphates and their oxidation products, all known to occur during degradation of [35S]SDTES by pure bacterial isolates. Triethylene glycol monosulphate was the major catabolite together with smaller amounts of di- and monoethylene glycol monosulphates implying, by analogy with pure cultures, that ether-cleavage was the major primary biodegradation step. The oxidation product (carboxylate derivative) of each glycol sulphate was also detected together with metabolites tentatively identified as omega-/beta-oxidation products of the dodecyl chain. Relatively little SO2-4 was liberated directly from SDTES but mixed cultures derived from sewage could metabolize the sulphated glycols to SO2-4. The environmental relevance of these degradation routes was established by following metabolite production from [35S]SDTES in full-scale river-water die-away tests. Triethylene glycol sulphate was formed first, then rapidly oxidized to acetic acid 2-(diethoxy sulphate) which persisted as the major metabolite for 2-3 weeks. Small amounts of sulphated derivatives of di- and monoethylene glycols were also detected during the same period. Very little SO2-4 was formed directly from SDTES but large amounts accompanied the eventual disappearance of glycol sulphate derivatives. None of the 35S-labelled organic metabolites was persistent and, whenever [35S]SDTES was a component of a commercial mixture, all ester sulphate was completely mineralized to 35SO4(2-) within 28 d.     

Microbial metabolism of 2-chlorophenol, phenol and rho-cresol by Rhodococcus erythropolis M1 in co-culture with Pseudomonas fluorescens P1

Chlorophenolic waste most often contains phenol and rho-cresol along with chlorophenols. A Rhodococcus erythropolis strain M1 was isolated with the ability to degrade 2-chlorophenol, phenol and p-cresol (100 mgl(-1), each) in 18, 24 and 20 h, respectively, with negligible lag. However, Rhodococcus sp. characterized by low growth rate, pose a threat to be outgrown by bacteria occurring in natural habitats. In the present study, interaction of R. erythropolis M1 with another isolated bacteria generally encountered in activated sludge for water treatment like Pseudomonas fluorescens P1 was studied. 2-chlorophenol, phenol and p-cresol were selected as the substrates for the study. Viable cell counts showed competitive interaction between the species on 2-chlorophenol and phenol. Specific growth rate of pure culture of R. erythropolis M1 was higher than P. fluorescens P1 on 2-chlorophenol. However, in mixed culture, P. fluorescens P1 showed higher growth rate. Degradation of phenol showed higher growth rate of R. erythropolis M1 both in pure and in mixed culture form. Degradation of p-cresol had shown similar counts for both populations indicating neutral type of interaction. This observation was substantiated by detecting the growth rate, where both cultures had similar growth rate in pure and in the mixed culture form. Rate of 2-chlorophenol degradation was higher when R. erythropolis M1 was used as the pure culture as compared to the degradation rates observed with the P. fluorescens P1 or with the mixed culture. However, in case of phenol and p-cresol, degradation by the mixed culture had resulted in higher degradation rates as compared to the degradation of the substrates by both the axenic cultures.     

Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [(35)S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic (35)SO(4) (2-) is liberated into the media. Cell extracts contain this anion as the major (35)S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[(35)S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic (35)SO(4) (2-) from sodium hexan-1-yl [(35)S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.     

The role of soil microbes in plant sulphur nutrition

Chemical and spectroscopic studies have shown that in agricultural soils most of the soil sulphur (>95%) is present as sulphate esters or as carbon-bonded sulphur (sulphonates or amino acid sulphur), rather than inorganic sulphate. Plant sulphur nutrition depends primarily on the uptake of inorganic sulphate. However, recent research has demonstrated that the sulphate ester and sulphonate-pools of soil sulphur are also plant-bioavailable, probably due to interconversion of carbon-bonded sulphur and sulphate ester-sulphur to inorganic sulphate by soil microbes. In addition to this mineralization of bound forms of sulphur, soil microbes are also responsible for the rapid immobilization of sulphate, first to sulphate esters and subsequently to carbon-bound sulphur. The rate of sulphur cycling depends on the microbial community present, and on its metabolic activity, though it is not yet known if specific microbial species or genera control this process. The genes involved in the mobilization of sulphonate- and sulphate ester-sulphur by one common rhizosphere bacterium, Pseudomonas putida, have been investigated. Mutants of this species that are unable to transform sulphate esters show reduced survival in the soil, indicating that sulphate esters are important for bacterial S-nutrition in this environment. P. putida S-313 mutants that cannot metabolize sulphonate-sulphur do not promote the growth of tomato plants as the wild-type strain does, suggesting that the ability to mobilize bound sulphur for plant nutrition is an important role of this species.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Enhancing Manganese Recovery from Low-Grade Ores by Using Mixed Culture of Indigenously Isolated Bacterial Strains

Conventional leaching methods for manganese (Mn) recovery require strong acids and are threatening to the environment. Alternatively, the use of microbes for Mn recovery is environment friendly in nature. The present investigation compares the capacity of pure and mixed cultures of native bacterial strains for bioleaching of low-grade Mn ores. The ability of the isolated microorganisms to recover Mn was evaluated in shake flasks for 20 days under optimized conditions of pulp density (2%), sucrose concentration (2 g/100 mL), initial pH 6.5, and 30°C incubation temperature. In pure culture form, Acinetobacter sp. MSB 5 (70%) was found to have a higher bioleaching potential than Lysinibacillus sp. MSB 11 (67%). Mixed culture of Acinetobacter sp. MSB 5 and Lysinibacillus sp. MSB 11 was found to perform better than the pure cultures with 74% extraction of Mn. The presence of mixed culture increased the dissolution rate and the recovery percentage of Mn. The respective growth pattern of the cultures was in synchronization to their Mn bioleaching performances. This study underlines the importance of mixed cultures and Mn solubilizing activity of native bacterial strains for efficient Mn biorecovery.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Comparison of microbial and photochemical processes and their combination for degradation of 2-aminobenzothiazole

The transformation of 2-aminobenzothiazole (ABT) was studied under various conditions: (i) a photodegradation process at a lambda of >300 nm in the presence of an Fe(III)-nitrilotriacetic acid complex (FeNTA), (ii) a biodegradation process using Rhodococcus rhodochrous OBT18 cells, and (iii) the combined processes (FeNTA plus Rhodococcus rhodochrous in the presence or absence of light). The transformation of ABT in the combined system, with or without light, was much more efficient (99% degradation after 25 h) than in the separated systems (37% photodegradation and 26% biodegradation after 125 h). No direct photolysis of ABT was observed. The main result seen is the strong positive impact of FeNTA on the photodegradation, as expected, and on the biotransformation efficiency of ABT, which was more surprising. This positive impact of FeNTA on the microbial metabolism was dependent on the FeNTA concentration. The use of UV high-performance liquid chromatography, liquid chromatography-electrospray ionization mass spectrometry, and in situ (1)H nuclear magnetic resonance provided evidence of the intermediary products and thus established transformation pathways of ABT in the different processes. These pathways were identical whether the degradation process was photo- or biotransformation. A new photoproduct was identified (4OH-ABT), corresponding to a hydroxylation reaction on position 4 of the aromatic ring of ABT.     

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.     

Impact of dust exposure on mixed bacterial cultures and during eukaryotic cell co-culture infections

On a daily basis, humans, and their colonizing microbiome, are exposed to both indoor and outdoor dust, containing both deleterious organic and inorganic contaminants, through dermal contact, inhalation, and ingestion. Recent studies evaluating the dust exposure responses of opportunistic pathogens, such as Escherichia coli and Pseudomonas aeruginosa, revealed significant increases in biofilm formation following dust exposure. In this study, the effects of dust exposure on mixed bacterial cultures as well as HT-29 co-cultures were evaluated. As it was observed in pure, single bacterial cultures earlier, neither indoor nor outdoor dust exposure (at concentrations of 100 μg/mL) influenced the growth of mixed bacterial liquid cultures. However, when in paired mixed cultures, dust exposure increased sensitivity to oxidative stress and significantly enhanced biofilm formation (outdoor dust). More specifically, mixed cultures (E. coli-Klebsiella pneumoniae, K. pneumoniae-P. aeruginosa, and E. coli-P. aeruginosa) exhibited increased sensitivity to 20 and 50 mM of H2O2 in comparison to their pure, single bacterial culture counterparts and significantly enhanced biofilm production for each mixed culture. Finally, bacterial proliferation during a eukaryotic gut cell (HT29) co-culture was significantly more robust for both K. pneumoniae and P. aeruginosa when exposed to both house and road dust; however, E. coli only experienced significantly enhanced proliferation, in HT29 co-culture, when exposed to road dust. Taken together, our findings demonstrate that bacteria respond to dust exposure differently when in the presence of multiple bacterial species or when in the presence of human gut epithelial cells, than when grown in isolation.     

Alkane utilization by Rhodococcus strain NTU-1 alone and in its natural association with Bacillus fusiformis L-1 and Ochrobactrum sp

Linear (n-hexadecane) and branched (pristane) alkanes were degraded by a mixed culture isolated from an oil-contaminated field. The degradation was accompanied by formation of biofloccules. The culture was composed of Rhodococcus strain NTU-1, Bacillus fusiformis L-1, and Ochrobactrum sp. Rhodococcus strain NTU-1 carried out the degradation of the alkane via a hydroxylase. Bacillus fusiformis L-1 and Ochrobactrum sp. did not degrade the alkanes but aided the flocculation by forming more rigid bacterial aggregates that enhanced the trapping of alkanes. In batch cultures, transformation and removal of the linear and branched alkanes was achieved within 66 h with more than 95% efficiency.     

Metabolism of select amino acids in bacteria from the pig small intestine

This study investigated the metabolism of select amino acids (AA) in bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum and ileum of pigs. Cells were incubated at 37°C for 3 h in anaerobic media containing 0.5–5 mM select AA plus [U-¹⁴C]-labeled tracers to determine their decarboxylation and incorporation into bacterial protein. Results showed that all types of bacteria rapidly utilized glutamine, lysine, arginine and threonine. However, rates of the utilization of AA by pure cultures of E. coli and Klebsiella sp. were greater than those for mixed bacterial cultures or Streptococcus sp. The oxidation of lysine, threonine and arginine accounted for 10% of their utilization in these pure bacterial cultures, but values were either higher or lower in mixed bacterial cultures depending on AA, bacterial species and the gut segment (e.g., 15% for lysine in jejunal and ileal mixed bacteria; 5.5 and 0.3% for threonine in jejunal mixed bacteria and ileal mixed bacteria, respectively; and 20% for arginine in ileal mixed bacteria). Percentages of AA used for bacterial protein synthesis were 50–70% for leucine, 25% for threonine, proline and methionine, 15% for lysine and arginine and 10% for glutamine. These results indicate diverse metabolism of AA in small-intestinal bacteria in a species- and gut compartment-dependent manner. This diversity may contribute to AA homeostasis in the gut. The findings have important implications for both animal and human nutrition, as well as their health and well-beings.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Isolation and physiological characterisation of Thiobacillus aquaesulis sp. nov., a novel facultatively autotrophic moderate thermophile

A moderately thermophilic, facultatively chemolithoautotrophic thiobacillus isolated from a thermal sulphur spring is described. It differs from all other species currently known to be in culture. It grows lithoautotrophically on thiosulphate, trithionate or tetrathionate, which are oxidized to sulphate. Batch cultures on thiosulphate do not produce tetrathionate, but do precipitate elemental sulphur during growth. In autotrophic chemostat cultures the organism produces yields on thiosulphate, trithionate and tetrathionate that are among the highest observed for a Thiobacillus. Autotrophic cultures contain ribulose bisphosphate carboxylase. Heterotrophic growth has been observed only on complex media such as yeast extract and nutrient broth. It is capable of autotrophic growth and denitrification under anaerobic conditions with thiosulphate and nitrate. It grows between 30 to 55° C, and pH 7 to 9, with best growth at about 43°C and pH 7.6. It contains ubiquinone Q-8, and its DNA contains 65.7 mol% G+C. The organism is formally described and named as Thiobacillus aquaesulis.Now the Department of Biological Sciences     

Biological sulphide oxidation in a fed-batch reactor

This study shows that, in a sulphide-oxidizing bioreactor with a mixed culture of Thiobacilli, the formation of sulphur and sulphate as end-products from the oxidation of sulphide can be controiledinstantaneously and reversibiy by the amount of oxygen supplied. It was found that at sulphide loading rates of up to 2.33 mmol7/L . h, both products can be formed already at oxygen concentrations below 0.1 mg/L. Because the microorganisms tend to form sulphate rather than forming sulphur, the oxygen concentration is not appropriate to optimize the sulphur production. Within less than 2 h, the system can be switched reversibly from sulphur to sulphate formation by adjusting the oxygen flow. This is below the minimum doubling time (2.85 h) of, e.g., Thiobacillus neapolitanus and Thiobacillus 0,(18) which indicates that one metabolic type of organism can probably perform both reactions. Under highly oxygen-limited circumstances, that is, at an oxygen/sulphide consumption ratio below 0.7 mol . h(-1) mol . h(-1) thiosulphate is abundantly formed. Because the chemical sulphide oxidation results mainly in the formation of thiosulphate, it is concluded that, under these circumstances, the biological oxidation capacity of the system is lower than the chemical oxidation capacity. The oxidation rate of the chemical sulphide oxidation can be described by a first-order process (k =-0.87 h(-1)).(c) 1995 John Wiley & Sons, Inc.