Aerobic Cr(VI) Reduction by Bacteria in Culture and Soil Conditions

We compared the performance of aerobic Cr(VI)-reducing bacteria isolated from Cr(VI)-contaminated soil in pure and mixed cultures of five isolated strains. The mixed culture had increased reduction rates compared to individual cultures. Cr(VI) reduction was observed in sterile soil inoculated with Pseudomonas fluorescens and in non-sterile soil with and without inoculation with P. fluorescens at initial pore water concentrations up to 1,600 mg Cr(VI)/L, whereas in culture the maximum inhibitory concentration was 500 mg Cr(VI)/L. Linear rates of Cr(VI) reduction in non-sterile soil amended with peptone were ～5 to 8 times higher than those observed in the mixed culture. Inoculation of non-sterile soil with P. fluorescens did not further enhance Cr(VI) reduction rates. Our results indicate that evaluation of Cr(VI) reduction capacity in Cr(VI)-contaminated soil for in-situ bioremediation purposes should not be done solely in pure culture. Although the latter may be used initially to assess the effects of process parameters (e.g., pH, temperature), the rate and extent of Cr(VI) reduction should be determined in soil for bioremediation design purposes.     

Response to oxygen of diazotrophic Azospirillum brasilense — Arthrobacter giacomelloi mixed batch culture

Azospirillum brasilense and Arthrobacter giacomelloi were grown together in batch culture under different oxygen pressures. The response to oxygen of growth, nitrogenase activity and respiration rate was determined. The two microorganisms were found to be able to coexist all over the range of partial oxygen pressures examined, that is from 0.004–0.20 bar. Nitrogenase activity by mixed culture of A. brasilense and A. giacomelloi always appeared higher than that of A. brasilense pure culture. Low respiratory activity at partial oxygen pressures higher than 0.02 bar by both pure and mixed cultures seemed not to account for the high nitrogenase activity and improved oxygen tolerance of the mixed culture.Abbreviations pO₂ partial oxygen pressure     

Dense growth of aerobic bacteria in a bench-scale fermentor

Escherichia coli B, Escherichia coli MRE 600, Escherichia coli K 12-3300, Pseudomonas fluorescens, and Aerobacter aerogenes were grown exponentially in a bench-scale fermentor to cell concentrations in the range of 20 to 41 g dry cells/liter at 30°C and 30 to 55 g dry cells/liter at 25°C. The high cell concentrations were achieved in a growth system previously described for growth of Escherichia coli W (Biotechnol. Bioeng., 16 , 933 (1974); ibid. 17 , 227 (1975)). Various enzyme activity levels in the high-concentration cells were compared to those in cells grown in conventional low-density cultures. No significant differences were found. The culture supernatants were found to be essentially free of high-molecular weight metabolic or cell lysis products. Yield constants for glucose, nitrogen, oxygen, and phosphorus were also determined in the dense cultures and some of their relations to the growth conditions are discussed.     

Survival in Sterile Soil and Atrazine Degradation of Pseudomonas sp. Strain ADP Immobilized on Zeolite

In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP.     

Iron Bound-Siderophores, Cyanic Acid, and Antibiotics Involved in Suppression of Thielaviopsis basicola by a Pseudomonas fluorescens Strain

A Pseudomonas fluorescens strain (CHAo) involved in suppression of black root rot caused by Thielaviopsis basicola in the field, inhibited T. basicola when colonizing roots grown under sterile conditions or when grown on culture, media. Under these conditions it produced siderophores (iron chelating compounds), cyanic acid, and several antibiotics. Iron-free siderophores inhibited neither the germination of endoconidia or chlamydospores nor the mycelial growth of T. basicola, but reduced the production of endoconidia. On the contrary, siderophores complexed with Fe³⁺ strongly inhibited mycelium growth and spore germination; free iron was less toxic than iron-bound siderophores. Therefore, contrary to what was believed to date, siderophores seem to be toxic not because they deplete iron but because they increase its concentration to the point where it becomes highly toxic. Cyanic acid and the antibiotics also inhibited the growth of T. basicola. Whetherall these compounds are involved in disease control in the soil remains, however, to be determined.     

Production of parathion hydrolase activity

Summary A mixed bacterial culture which was obtained in a previous enrichment grew on parathion, an organophosphate insecticide, as a sole carbon and energy source. A cell-free enzyme preparation from this culture detoxified by hydrolysis eight commercially used organophosphate insecticides.Fermentation procedures for the production of this parathion hydrolase activity were examined to determine if this enzyme activity could be produced economically. The mixed culture was grown using sterile or non-sterile procedures in 4 or 11 continuous and batch culture fermentations. A pure Pseudomonas sp isolated from the mixed culture expressed parathion hydrolase activity when grown under axenic fermentation conditions on industrially used media such as meat extract, soya bean meal, and corn extract. The optimal conditions for production of parathion hydrolase activity were determined for both pure and mixed cultures. The yield of parathion hydrolase activity/ of fermentation broth per hour was improved 22 fold by growing the pure culture on an industrial meat extract medium instead of the mixed culture on parathion.     

The Influence of <Emphasis Type="Italic">P. fluorescens</Emphasis> Cell Morphology on the Lytic Performance and Production of Phage ϕIBB-PF7A

This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage ϕIBB-PF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25°C and also at 30°C under static conditions, and elongated morphology only at 30°C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 μm longer than rod cells. Rod-shaped hosts were best infected by phages at 25°C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P > 0.05) to those obtained at the optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25°C (the optimal temperature) and 30°C, respectively. Phage adsorption was faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.     

Effect of tungsten and molybdenum on growth of a syntrophic coculture of <Emphasis Type="Italic">Syntrophobacter fumaroxidans</Emphasis> and <Emphasis Type="Italic">Methanospirillum hungatei</Emphasis>

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H₂/CO₂ required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H₂ as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.     

Intraspecific competition as a regulating factor limiting the number of transconjugants in soil

A laboratory study was carried out to determine survival of transconjugant cells ofPseudomonas fluorescens intro duced into sterile soil. The transconjugant survived significantly better when it was the only strain inoculated into the soil; when introduced into soil pre-colonized by the recipient strain, the transconjugant was undetectable. These results indicate that intraspecific competition is a regulating factor limiting the number of transconjugants in soil.     

Efficacy of Pseudomonas fluorescens and Paecilomyces lilacinus against Meloidogyne graminicola infecting rice under system of rice intensification

The talc-based formulations of the plant growth promoting rhizobacterium, Pseudomonas fluorescens and egg parasitic fungi, Paecilomyces lilacinus, were evaluated as seed treatment, soil application and combination of both for the management of M. graminicola in fields of rice grown under system of rice intensification. Both the bioformulations significantly reduced the root invasion and soil populations of M. graminicola but P. fluorescens was most effective when applied as seed cum soil application and seed treatment alone. Effect of these treatments was comparable with the standard chemical carbofuran application. The introduced P. fluorescens survived significantly in rice roots when applied as seed cum soil application and seed application alone than as soil application. There was significant increase in phenol, peroxidase and chitinase accumulation in plants treated with P. fluorescens. Application of bioagents had positive influence on growth parameters such as plant height, root length, shoot weight, root weight and number of tillers per hill. Application of P. fluorescens as seed cum soil treatment resulted in higher grain yield, which was 20.6%–26.9% increase over control followed by P. fluorescens as seed treatment alone that increased grain yield of rice by 10.7%–11.2% than control. However, economic returns per investment was higher when P. fluorescens was applied as seed treatment alone (1:8.8–1:12.0 incremental cost benefit ratio) followed by the P. fluorescens as seed cum soil treatment (1:6.2–1:9.7 incremental cost benefit ratio).     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Volatile Ketone Formation in Bacteria: Release of 3-Oxopentanoate by Soil Pseudomonads During Growth on Heptanoate

During enrichments to search for soil bacteria that form the volatile ketones, acetone and butanone, we isolated pure cultures of Pseudomonas aureofaciens, P. fluorescens, and P. putida that excrete the β-keto-acid, 3-oxopentanoate (3-OPA), when grown on heptanoic acid as carbon source. Analysis of 3-OPA used enzymatic decarboxylation by acetoacetate decarboxylase to yield butanone, which was detected by headspace gas chromatography. The formation of 3-OPA was strongly dependent on heptanoic acid concentration, the level of oxygen, and the state of growth, and was not seen with even-chain or other odd-chain fatty acids. Uptake of 3-OPA during stationary phase of growth is probably related to polyhydroxyalkanoate (PHA) formation in these isolates. A model for formation and release of 3-OPA is proposed. Received: 28 August 2000 / Accepted: 2 October 2000     

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech     

The distribution of acetohydroxyacid synthase in soil bacteria

Most bacteria possess the enzyme acetohydroxyacid synthase, which is used to produce branched-chain amino acids. Enteric bacteria contain several isozymes suited to different conditions, but the distribution of acetohydroxyacid synthase in soil bacteria is largely unknown. Growth experiments confirmed that Escherichia coli, Salmonella enterica serotype Typhimurium, and Enterobacter aerogenes contain isozymes of acetohydroxyacid synthase, allowing the bacteria to grow in the presence of valine (which causes feedback inhibition of AHAS I) or the sulfonylurea herbicide triasulfuron (which inhibits AHAS II) although a slight lag phase was observed in growth in the latter case. Several common soil isolates were inhibited by triasulfuron, but Pseudomonas fluorescens and Rhodococcus erythropolis were not inhibited by any combination of triasulfuron and valine. The extent of sulfonylurea-sensitive acetohydroxyacid synthase in soil was revealed when 21 out of 27 isolated bacteria in pure culture were inhibited by triasulfuron, the addition of isoleucine and/or valine reversing the effect in 19 cases. Primers were designed to target the genes encoding the large subunits (ilvB, ilvG and ilvI) of acetohydroxyacid synthase from available sequence data and a ∼355 bp fragment in Bacillus subtilis, Arthrobacter globiformis, E. coli and S. enterica was subsequently amplified. The primers were used to create a small clone library of sequences from an agricultural soil. Phylogenetic analysis revealed significant sequence variation, but all 19 amino acid sequences were most closely related to published large subunit acetohydroxyacid synthase amino acid sequences within several phyla including the Proteobacteria and Actinobacteria. The results suggested the majority of soil microorganisms contain only one functional acetohydroxyacid synthase enzyme sensitive to sulfonylurea herbicides.     

Development of a method for the application of solid-phase microextraction to monitor biodegradation of volatile hydrocarbons during bacterial growth on crude oil

A quantitative solid-phase microextraction, gas chromatography, flame ionization detector (SPME-GC-FID) method for low-molecular-weight hydrocarbons from crude oil was developed and applied to live biodegradation samples. Repeated sampling was achieved through headspace extractions at 30°C for 45 min from flasks sealed with Teflon Mininert. Quantification without detailed knowledge of oil–water–air partition coefficients required the preparation of standard curves. An inverse relationship between retention time and mass accumulated on the SPME fibre was noted. Hydrocarbons from C₅ to C₁₆ were dated and those up to C₁₁ were quantified. Total volatiles were quantified using six calibration curves. Biodegradation of volatile hydrocarbons during growth on crude oil was faster and more complete with a mixed culture than pure isolates derived therefrom. The mixed culture degraded 55% of the compounds by weight in 4 days versus 30–35% by pure cultures of Pseudomonas aeruginosa, Rhodococcus globerulus or a co-culture of the two. The initial degradation rate was threefold higher for the mixed culture, reaching 45% degradation after 48 h. For the mixed culture, the degradation rate of individual alkanes was proportional to the initial concentration, decreasing from hexane to undecane. P. fluorescens was unable to degrade any of the low-molecular-weight hydrocarbons and methylcyclohexane was recalcitrant in all cases. Overall, the method was found to be reliable and cost-effective. Journal of Industrial Microbiology & Biotechnology (2000) 25, 155–162. Received 04 March 2000/ Accepted in revised form 25 June 2000     

Siderophore mediated plant growth promotion at low temperature by mutant of fluorescent pseudomonad⋆

A cold resistant mutant of Pseudomonas fluorescens ATCC 13525 was developed, which could grow equally well at 25 and 10 °C and its effect on plant growth promotion under in vitro and in situ conditions was observed. Siderophore estimation revealed it to be a siderophore-overproducing mutant (17-fold increase) when compared to its wild type counterpart. A gnotobiotic root elongation assay indicated that the mutant (CRPF₉) promoted growth more than its wild type both at 25 and 10 °C, indicating its effectiveness at low temperature. Further, root colonization studies showed that CRPF₉ was an efficient rhizosphere colonizer, inducing a significant increase in root (35%) and shoot length (28%) of mung bean plants in unsterilized soil system. The persistence and stability of the mutant was evident in rhizospheric soil. A sand culture experiment showed that ferric citrate was better than Fe(OH)₃ as an iron source for plant growth, but in the presence of CRPF₉ both salts were comparable. This study demonstrates the potential of chemical mutagenesis for improving the plant growth promoting properties of a P. fluorescens strain and its stimulating impact on plant growth promotion at low temperature both under in vitro and in situ conditions.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.     

H2 production by mixed cultures

Production of H₂ from glucose by an anoxygenic phototrophic bacterium (Rhodobacter sphaeroides), a cyanobacterium (Synechococcus cedrorum) and a heterotrophic bacterium (Pseudomonas fluorescens) was tested individually and in mixed cultures of various combinations in light. H₂ production was maximal with a mixed culture of R. sphaeroides and P. fluorescens, which could be further enhanced by immobilization of the bacteria in alginate gel. Inhibition of H₂ photoproduction was observed in a mixture of S. cedrorum and P. fluorescens and a co-culture of all the three organisms.Ch. Sasikala and Ch. V. Ramana are and G. S. Prasad was with the Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad-500 007, India. G. S. Prasad is now with the Microbial Type Culture Collection Centre (MTCC), IMTECH, Chandigar, India.     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.