Indigenous Heterotrophic Bacteria Research Articles

Biostimulation Potential of Nypa fruticans Ash on Indegenous Heterotrophic Bacteria and Fungi for Enhanced Remediation of Hydrocarbon Polluted Soil at B. Dere, Rivers State, Nigeria

This study was designed to evaluate the biostimulation potential of Nypa feuticans ash on the growth of indigenous heterotrophic bacteria and fungi for enhanced remediation of hydrocarbon polluted soil at B. Dere, Rivers State, Nigeria in an ex-situ experiment, using the completely randomized split plot experimental design. 2kg of oil polluted soils were treated with 10g, 20g, 30g, 40g, 50g and 60g of Nypa fruticans ash and monitored for biostimulation and growth of indigenous biodegrading heterotrophic bacteria (THBC) and fungi (THFC). The results of four months post residual and cumulative effect on treated soil showed optimum improvement of microbial load at 60g/2kg treatment of Nypa fruticans ash. Findings on THBC for post residual treatment soil ranged from 0.1×10⁵ to 8.0 ×10⁴ cfu/g, while the THFC ranged from 0.1×10⁴ to 3.0 ×10⁴ cfu/g. THBC for post cumulative treated soil ranged from 2.7 ×10⁵ to 9.0 ×10⁴ cfu/g, while THFC ranged from 0.2 × 10⁴ to 3.0 ×10⁴ cfu/g. Three bacteria genera isolated from the post treated soil ware Bacillus spp. (36.3%), Clostridium spp (33.4%) and staphylococcus spp. (30.3%), while two fungi genera namelyYeast spp. (58%) and Muccor spp. (42%) were isolated from the post treated soil. The high-count THB and THF recorded in both residual and cumulative treated soil compared to the oil polluted untreated soil is attributed to Nypa fruticans ash capacity to neutralize the toxic effects of the crude oil on the microbial population by rapid improvement of the soil physiochemical properties, favoring the growth of indigenous microorganisms in the soil. This study has shown that treatment of crude oil polluted soil with Nypa fruticans ash at 60g/2kg showed optimum improvement which enhanced the biostimulation and growth of indigenous petroleum degrading microorganism.

Biotransformation and removal of arsenic oxyanions by Alishewanella agri PMS5 in biofilm and planktonic states

Arsenic oxyanions are toxic chemicals that impose a high risk to humans and other living organisms in the environment. The present study investigated indigenous heterotrophic bacteria in the tailings dam effluent (TDE) of a gold mining factory. Thirty-seven arsenic resistant bacteria were cultured on Reasoner's 2A agar supplemented with arsenic salts through filtration. One strain encoded as PMS5 with the highest resistance to 140-mM sodium arsenite and 600-mM sodium arsenate in tryptic soy broth was selected for further investigations. According to phenotypic examinations and 16S rDNA sequence analysis, PMS5 belonged to the genus Alishewanella and was sensitive to most of the examined antibiotics. The biosorption and bioaccumulation abilities of arsenic salts were observed in this isolate based on Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) and biosorption and bioaccumulation data. PMS5 was also found to cause the volatilization and biotransformation of arsenic oxyanions through their oxidation and reduction. Moreover, the contribution of PMS5 to arsenic (3+, 5+) bioprocessing under oligotrophic conditions was confirmed in fixed-bed reactors fed with the TDE of the gold factory (R1) and synthetic water containing As5+ (R2). According to biofilm assays such as biofilm staining, cell count, detachment assay and SEM, the arsenic significantly reduced the biofilm density of the examined reactors compared to that of the control (R3). Arsenate reduction and arsenite oxidation under bioreactor conditions were respectively obtained as 75.5–94.7% and 8%. Furthermore, negligible arsenic volatilization (1.2 ppb) was detected.

Determination of log removal values of bacteria by spiral-wound reverse osmosis modules and a hollow fiber ultrafiltration module using Escherichia coli and indigenous heterotrophic bacteria as indicators.

The use of reverse osmosis (RO) membranes has been expanding not only to medical applications but also to water supply and reclaimed water applications due to its strong ability to remove a wide range of contaminants. Many researchers reported RO performance as a barrier against waterborne viruses; however, there are limited reports on its ability to remove bacteria from water. This investigation evaluated the removal performances of several spiral-wound RO modules and a hollow fiber ultrafiltration (UF) module in two different ways: dosing tests in batch-wise mode operation and in continuous-mode operation. The dosing tests of Escherichia coli using RO modules confirmed that E. coli could leak from the feed-side into the permeate. The log removal values (LRVs) (4.21- to >7.44-log10) by the RO modules from different production lots were found to vary greatly. In continuous-mode operation of the RO module, the LRVs for indigenous heterotrophic bacteria decreased over the operation period. These results clearly illustrate that bacteria, which originate on the feed-side, can leak into the permeate-side and then begin to proliferate in the permeate. On the other hand, using a UF module, E. coli was not detected in the permeate regardless of the operation mode.

Optimization of manganese bioleaching activity and molecular characterization of indigenous heterotrophic bacteria isolated from the sulfuric area

Abstract. Prasidya DA, Wilopo W, Warmada IW, Retnaningrum E. 2019. Optimization of manganese bioleaching activity and molecular characterization of indigenous heterotrophic bacteria isolated from the sulfuric area. Biodiversitas 20: 1904-1909. The present research evaluated the manganese bioleaching potency of a heterotrophic bacteria KB3B1. This bacterial strain has been isolated from sulfuric area located at Ungaran, Middle of Java, Indonesia using modified 9K medium by adding of several organic nutrients. The manganese bioleaching activities of the strain was analysed by applying of varying glycine concentrations (0, 5, 10, 15 mg mL-1) with pyrolusite pulp densities of 0.02 g cm-3 on a rotary shaker at 180 rpm for 18 days incubation. Several parameters, including the growth of bacteria, pH values, the concentration of soluble manganese and cyanide, were investigated at the interval of 3 days. Molecular characteristics of the strain were further analyzed based on 16S rDNA gene sequences. After 15 days, the maximum yield of manganese 16.6% was achieved under the addition of 10 mg mL-1 glycine. This maximum extract obtained was followed by the maximum bacterial growth, pH, and cyanide product of the strain. Phylogenetic analysis showed that the strain was closely related with Bacillus niacini EP89. Besides, the average frequencies of guanine and cytosine (G+C) of the strain was in same range as that of the reference bacteria in the GenBank and Bergey's Manual Systematics of Bacteria.

Metal Leaching and Reductive Dissolution of Iron from Contaminated Soil and Sediment Samples by Indigenous Bacteria and Bacillus Isolates

ABSTRACTThe purpose of this study was to leach Cu, Zn, As, and Fe from contaminated soil and sediment samples with indigenous heterotrophic bacteria isolated from the study sites. The sediment contained Fe in the form of goethite and low concentrations of other metals. The soil contained hematite and high concentrations of other metals. The environmental conditions affected the bacterial activity in the metals dissolution. As and Fe were the major metals leached from the sediment sample while a minor fraction of Cu was solubilized. Cu and Zn were the major metals leached from the soil sample while only a minor fraction of Fe was dissolved. As a control, a disinfectant was used for partial inactivation of indigenous bacteria. This treatment had a negative effect on the leaching of Fe, Zn and As from soil and sediment samples, but it increased Cu dissolution from the sediment. Bacterial different dissolution of Fe during soil and sediment bioleaching was also investigated with ferrihydrite. The iron concentration was much higher during ferrihydrite dissolution when indigenous bacteria from sediment were used compared to indigenous bacteria isolated from soil. The indigenous bacterial inoculum provided more biological and metabolic diversity which may account for the difference in reductive iron reduction from ferrihydrite. The Bacillus cultures isolated from soil and sediment samples showed similar efficiencies in reductive dissolution of ferrihydrite. The synergetic bacterial inhibition effect created by the environmental conditions can influence bioremediation effect.

Interactions of the metal tolerant heterotrophic microorganisms and iron oxidizing autotrophic bacteria from sulphidic mine environment during bioleaching experiments

Iron and sulfur oxidizing chemolithoautotrophic acidophilic bacteria, such as Acidithiobacillus species, hold the dominant role in mine environments characterized by low pH values and high concentrations of reduced sulfur and iron compounds, such as ores, rocks and acid drainage waters from mines. On the other hand, heterotrophic microorganisms, especially their biofilms, from these specific niches are receiving increased attention, but their potential eco-physiological roles have not been fully understood. Biofilms are considered a threat to human health, but biofilms also have beneficial properties as they are deployed in waste recycling and bioremediation systems. We have analyzed interactions of the metal tolerant heterotrophic microorganisms in biofilms with iron oxidizing autotrophic bacteria both from the sulphidic mine environment (copper mine Bor, Serbia). High tolerance to Cu2+, Cd2+ and Cr6+ and the presence of genetic determinants for the respective metal tolerance and biofilm-forming ability was shown for indigenous heterotrophic bacteria that included strains of Staphylococcus and Rhodococcus. Two well characterized bacteria- Pseudomonas aeruginosa PAO1 (known biofilm former) and Cupriavidus metallidurans CH34 (known metal resistant representative) were also included in the study. The interaction and survivability of autotrophic iron oxidizing Acidithiobacillus bacteria and biofilms of heterotrophic bacteria during co-cultivation was revealed. Finally, the effect of heterotrophic biofilms on bioleaching process with indigenous iron oxidizing Acidithiobacillus species was shown not to be inhibitory under in vitro conditions.

Rock weathering by indigenous heterotrophic bacteria of Bacillus spp. at different temperature: a laboratory experiment

The bio-weathering of basalt, granite and gneiss was experimentally investigated in this study. These rock-forming minerals weathered more rapidly via the ubiquitous psychrotrophic heterotrophic bacteria. With indigenous bacteria of Bacillus spp. from sediments of Lake Baikal, we traced the degradation process of silicate minerals to understand the weathering processes occurring at the change temperature in the subsurface environment with organic input. The bacteria mediated dissolution of minerals was monitored with solution and solid chemistry, X-ray analyses as well as microscopic techniques. We determined the impact of the bacteria on the mineral surface and leaching of K, Ca, Mg, Si, Fe, and Al from silicate minerals. In the samples the release of major structural elements of silicates was used as an overall indicator of silicate mineral degradation at 4°C and 18°C from five medium exchanges over 255 days of rock bioleaching. The increase of temperature importantly affected the efficiency of Fe extraction from granite and basalt as well as Si extraction from granite and gneiss. In comparison with elemental extraction order at 4°C, Ca was substituted first by Fe or Si. It is evident that temperature influences rock microbial weathering and results in a change of elements extraction.

Bioleaching of clays and iron oxide coatings from quartz sands

Silica sands of low quality contain free iron oxides, clay and silt. These impurities may be removed by biological leaching as well as by industrial washing. The fine fractions obtained from industrial washing of quartz sand from Šastín, Slovakia, with a higher content of iron-bearing minerals and clay minerals were used to study bioleaching of impurities of the quartz sands. The influence of biological leaching by indigenous heterotrophic bacteria and Bacillus spp. bacteria was evaluated by changes in chemical composition of the fine fractions before and after reaction. The bioleaching experiments were performed for 63days at 24°C. The extracted iron and clay products were characterized by XRD, FTIR, Mössbauer spectroscopy, chemical and granulometric analyses, SEM, EDX, and nitrogen adsorption techniques. The bacteria assisted in releasing the poorly ordered clay minerals from the surface of quartz particles via dissolution of amorphous Fe oxyhydroxides.

Molecular cloning of Brevundimonas diminuta for efficacy assessment of reverse osmosis devices

Brevundimonas diminuta is the test organism specified in the United States Environmental Protection Agency's (USEPA) reverse osmosis (RO) treatment device verification protocol. As non-selective growth medium is employed, enumeration of B. diminuta may be impaired due to interference by indigenous heterotrophic bacteria. Thus the microbial removal capability of the filtration system may be incorrectly assessed. As these treatment devices are used in emergency situations, the health of the public could be compromised. The objective of this study was to develop selective approaches for enumerating viable B. diminuta in test water. Two molecular approaches were investigated: expression of a kanamycin resistance gene and expression of a fluorescent protein gene. The USEPA protocol specifies a 0.3 μm cell size, so the expression of the selective markers were assessed following growth on media designed to induce this small cell diameter. The kan(R) strain was demonstrated to be equivalent to the wild type in cell dimension and survival following exposure to the test water. The kan(R) strain showed equivalent performance to the wild type in the RO protocol indicating that it is a viable alternative surrogate. By utilizing this strain, a more accurate validation of the RO system can be achieved.

Bioweathering of Kupferschiefer black shale (Fore-Sudetic Monocline, SW Poland) by indigenous bacteria: implication for dissolution and precipitation of minerals in deep underground mine

The Upper Permian polymetallic, organic-rich Kupferschiefer black shale in the Fore-Sudetic Monocline is acknowledged to be one of the largest Cu-Ag deposits in the world. Here we report the results of the first study of bioweathering of this sedimentary rock by indigenous heterotrophic bacteria. Experiments were performed under laboratory conditions, employing both petrological and microbiological methods, which permitted the monitoring and visualization of geomicrobiological processes. The results demonstrate that bacteria play a prominent role in the weathering of black shale and in the biogeochemical cycles of elements occurring in this rock. It was shown that bacteria directly interact with black shale organic matter to produce a widespread biofilm on the Kupferschiefer shale surface. As a result of bacterial activity, the formation of pits, bioweathering of ore and rock-forming minerals, the mobilization of elements and secondary mineral precipitation were observed. The chemistry of the secondary minerals unequivocally demonstrates the mobilization of elements from minerals comprising Kupferschiefer. The redistribution of P, Al, Si, Ca, Mg, K, Fe, S, Cu and Pb was confirmed. The presence of bacterial outer membrane vesicles on the surface of black shale was observed for the first time. Biomineralization reactions occurred in both the membrane vesicles and the bacterial cells.

Preozonation of primary‐treated municipal wastewater for reuse in biofuel feedstock generation

AbstractThe results of a laboratory scale investigation on ozone pretreatment of primary‐treated municipal wastewater for potential reuse in fermentation processes for the production of biofuels and bio‐based feedstock chemicals were presented. Semi‐batch preozonation with 3.0% (w/w) ozone at 1 L min−1 resulted into a considerable inactivation of the indigenous heterotrophic bacteria in the wastewater with less than 0.0002% comprising the ozone‐resistant fraction of the microbial population. The disinfection process was modeled using first‐order inactivation kinetics with a rate constant of 4.39 × 10−3 s−1. Chemical oxygen demand (COD) levels were reduced by 30% in 1‐h experiments. COD depletion was also modeled using a pseudo‐first‐order kinetics at a rate constant of 9.50 × 10−5 s−1. Biological oxygen demand (BOD5) values were reduced by 60% up to 20 min of ozonation followed by a plateau and some slight increases attributed to partial oxidation of recalcitrant materials. Ozone also had no substantial effect on the concentration of ammonium and phosphate ions, which are essential for microbial growth and metabolism. Preliminary tests indicated that oleaginous microorganisms could be cultivated in the ozonated wastewater, resulting in relatively higher cell densities than in raw wastewater and comparable results with autoclave‐sterilized wastewater. This process could potentially produce significant quantities of oil for biofuel production from municipal wastewater streams. © 2010 American Institute of Chemical Engineers Environ Prog, 2010

Selective Enhancement of the Fluorescent Pseudomonad Population After Amending the Recirculating Nutrient Solution of Hydroponically Grown Plants with a Nitrogen Stabilizer

Fluorescent pseudomonads have been associated, via diverse mechanisms, with suppression of root disease caused by numerous fungal and fungal-like pathogens. However, inconsistent performance in disease abatement, after their employment, has been a problem. This has been attributed, in part, to the inability of the biocontrol bacterium to maintain a critical threshold population necessary for sustained biocontrol activity. Our results indicate that a nitrogen stabilizer (N-Serve, Dow Agrosciences) selectively and significantly enhanced, by two to three orders of magnitude, the resident population of fluorescent pseudomonads in the amended (i.e., 25 microg ml(-1) nitrapyrin, the active ingredient) and recycled nutrient solution used in the cultivation of hydroponically grown gerbera and pepper plants. Pseudomonas putida was confirmed as the predominant bacterium selectively enhanced. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA suggested that N-Serve selectively increased P. putida and reduced bacterial diversity 72 h after application. In vitro tests revealed that the observed population increases of fluorescent pseudomonads were preceded by an early growth suppression of indigenous aerobic heterotrophic bacteria (AHB) population. Interestingly, the fluorescent pseudomonad population did not undergo this decrease, as shown in competition assays. Xylene and 1,2,4-trimethylbenzene (i.e., the inert ingredients in N-Serve) were responsible for a significant percentage of the fluorescent pseudomonad population increase. Furthermore, those increases were significantly higher when the active ingredient (i.e., nitrapyrin) and the inert ingredients were combined, which suggests a synergistic response. P. putida strains were screened for the ability to produce antifungal compounds and for the antifungal activity against Pythium aphanidermatum and Phytophthora capsici. The results of this study suggest the presence of diverse mechanisms with disease-suppressing potential. This study demonstrates the possibility of using a specific substrate to selectively enhance and maintain desired populations of a natural-occurring bacterium such as P. putida, a trait considered to have great potential in biocontrol applications for plant protection.

Influence of introduced potential biocontrol agents on maize seedling growth and bacterial community structure in the rhizosphere

Two species of Pseudomonas chromosomally tagged with gfp, which had shown antagonistic activity against the tomato pathogen Ralstonia solanacearum in a previous study, were assessed for their impact in the rhizosphere of maize. Plant growth characteristics, numbers of indigenous heterotrophic bacteria, changes in the bacterial community structure according to the r/K strategy concept, and shifts in MIDI-FAME profiles of culturable bacterial fractions as well as total rhizosphere microbial communities were determined in relation to seed and soil treatment with the exogenous pseudomonads. The maize rhizosphere proved to be a suitable habitat for the introduced P. chlororaphis IDV1 and P. putida RA2, which showed good survival after introduction. However, both inoculants showed a small growth-reducing effect towards maize, which might have been caused by the high densities of inoculants used (i.e. competition for nutrients and action of metabolites produced) and/or changes in microbial community structure (both culturable bacterial fraction and the total microflora). Probably, an altered balance among the indigenous maize rhizosphere populations occurred. Thus, the culturable bacteria, as well as the total microflora in the rhizosphere, changed in response to the introduced pseudomonads, and their development was dependent on the growth stage of the plant. The FAME analyses showed that these microbial communities comprised different populations, and were separated according to, first, the method used (direct versus cultivation-based), second, sampling time, and, finally, inoculation level.

Modeling Bacterial Growth in Drinking Water: Effect of Nutrients

This article presents a model of growth of naturally occurring heterotrophic bacteria in the bulk water phase in the absence of disinfectant. The model considers growth with carbon, phosphorus, and nitrogen balance, death and lysis of bacteria, and conversion of less biodegradable organic carbon to assimilable organic carbon. Experimental data from two raw and two treated waters were used to test the model. The model describes the increase of live and dead bacterial cells in the water phase, and its output closely matches the experimental data. Such a model has the ability to characterize water nutrient status as well as to predict behavior of indigenous heterotrophic bacteria. The ability to predict bacterial population dynamics with respect to nutrients is beneficial for water treatment optimization. The model, based on microbiological measurements, helps to characterize treated water quality and project performance in terms of water quality into a distribution system.

Treatability Testing of Intrinsic Bioremediation, Biostimulation, And Bioaugmentation of Diesel-oil Contaminated Soil At 5 ° C

Abstract To support the remediation design for a diesel-oil pipeline break near a remote railway river crossing near the British Columbia and Alaska border, a treatability test was designed to assess the in-situ bioremediation potential under four conditions of soil amendments at 5 ° C. The four conditions were:intrinsic bioremediation, where the soil received only water;biostimulation with one application of slow-release fertilizer;bioaugmentation with one application of both fertilizer and a psychrotrophic (cold-adapted), hydrocarbon-degrading bacterial culture; andsurfactant enhanced bioavailability, where the soil received one application of fertilizer and treatment with a biodegradable surfactant solution. Tests were monitored by soil respiration and bacterial enumeration and total extractable hydrocarbon (TEH) content by GC/FID. After 40 days incubation under aerobic conditions, all tests showed significant reduction in diesel range (C7 - C30) THE contamination. The intrinsic control was least effective, but still showed 66% TEH reduction at 5 ° C. The biostimulated soil performed the best, allowing 86% TEH reduction. The bioaugmented soil and surfactant treated soil both allowed about 75% TEH reduction. The study concluded that simple biostimulation with slow-release fertilizers would be the most appropriate bioremediation approach for this site. Introduction A four-inch diameter petroleum pipeline, located adjacent to the rail tracks of the White Pass and Yukon Route Railroad, was broken by heavy equipment working in the area in October 1994. A significant quantity of Arctic diesel fuel leaked and immediate efforts were made to recover most of the product through the summer of 1995. Following site investigation in November 1995, a plume of diesel contamination was delineated in the gravelly cobbles and boulder fill supporting the railway bridge over the East Fork Skagway River. The plume had migrated downward through the fill to bedrock surface. Liquid product at the toe of the slope could discharge with shallow groundwater flow over ground surface into the river. The estimated 500 m3 of residual hydrocarboncontaminated fill supporting the railway bed required remediation to prevent continued contamination of the water passing through the contaminated area. Initial soil chemical and biological data indicated that active natural biodegradation of petroleum hydrocarbons may be occurring at the site, but appears limited by natural nutrient level and the indigenous bacterial population at the site. The gravelly soil near the toe of the slope contains between 1,000 and 5,000 mg/kg of total extractable hydrocarbons (TEH) and has an indigenous heterotrophic bacteria population of between 104 and 106 bacteria/ g of soil. Excavation of the slope and structural fill supporting the operating railway was not feasible; therefore, in-situ remediation options were considered. Because of the generally low volatility of some components of Arctic diesel and the lack of electrical power at the remote site, in-situ bioremediation was considered the most feasible option. To determine the optimal conditions for in-situ bioremediation of this hydrocarbon-contaminated soil, a laboratory-scale treatability study was carried out. Because of the cold climate near the Alaska-British Columbia border, it was imperative that the testing be performed at the expected ambient soil temperature of about 5 to 8 ° C.

Impact of introduced pseudomonas fluorescens mutants on indigenous rhizosphere microflora of bean

Mutants of Pseudomonas fluorescens were used for inoculation of bean (Phaseolus vulgaris L.) at different growth stages to assess their impact on indigenous culturable heterotrophic bacteria in the rhizosphere. Survival of inoculants, changes in numbers of the indigenous bacteria and their physiological state, and mi‐crobial community structures were determined using a simple agar plating method. The results showed that survival of introduced P. fluorescens mutants varied according to the growth stage of bean inoculated. However, overall, the mutants survived better in the rhizosphere with time, when they were inoculated on seeds. In contrast to some authors, the effect of mutants introduced was observed in the case of indigenous bacterial numbers and microbial community structures. Overall, no significant treatment effect was observed, when changes in values of kinetic parameters of colony formation were considered. Generally, cfu numbers, physiological state and community structure of indigenous microflora in the rhizosphere were depended on the effect of the interaction between the inoculants, the time after the introduction and the growth stage of plant.

Isolation and characterisation of the marine ultramicrobacteriumSphingomonassp. strain RB2256

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881–891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a ‘model oligotroph’ and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable ‘low-nutrient-conditioned’ phenotype.

Survival, plasmid transfer and impact of pseudomonas fluorescens introduced into soil

The survival of donor, recipient and transconjugant strains of Pseudomonas fluo‐rescens, transfer of plasmid RP4 from donor to recipient, and impact of the introduced strains on indigenous microflora was determined in a sandy‐loam soil. The introduced donor and recipient strains survived significantly better in sterile than in non‐sterile soil where a progressive decline in their numbers with time was observed. In both soils, conjugation and plasmid RP4 transfer from donor to recipient cells was observed during the first 3 days. 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. The introduced strains caused stimulation of total number of indigenous heterotrophic bacteria and selected physiological groups such as proteolytic, amylolytic and am monifying bacteria, but had no effect on the number of indigenous fungi. These results confirm the fact of limited survival of genetically modified pseudomonads introduced into soil. However, their potential for conjugation and impact on soil microflora are noteworthy from the aspect of ecological risk assessment.

Enteric virus removal/inactivation by coal-based media

Four coal-based media, viz. alum-pretreated or ferric hydroxide-impregnated Giridih bituminous coal and lignite (alum-GBC, FeGBC, alum-lignite and FeLignite) were laboratory tested to assess their potential in removing/inactivating enteric viruses in water. Batch-sorption screening tests, employing a poliovirus-spiked (3.10 × 10 7−1.49 × 10 8 PFU 1 −1) canal water, indicated high poliovirus sorption by FeGBC (7.07 × 10 6 PFU g −1) and alum-GBC (5.70 × 10 6 PFU g −1) in a short contact time of 5 min. Based on the results of further batch-sorption tests, using silver-incorporated media (alum/AgGBC, alum-GBCAg and FeGBCAg), as well as aesthetic water quality consideration and previous findings on removal of coliforms and turbidity, alum/AgGBC, alum-GBC and alum-GBCAg were included in downflow column studies (bed depth 10 cm; flow rate 99 ml h −1; bed flowthrough time 4.76 min), employing poliovirus-spiked (1.95 × 10 5−2.68 × 10 7 PFU 1 −1) canal water. All three media showed potential in removing/inactivating enteric viruses. In a separate column study (bed depth 25.4 cm; flow rate 500 ml h −1; flowthrough time 7.7 min), employing a joint challenge of poliovirus and rotavirus (2.47 × 10 7 PFU 1 −1), alum/AgGBC removed 59.3–86.5% of the viruses along with more than 99% reduction in indigenous heterotrophic bacteria. Alum/silver-pretreated bituminous coal medium appears promising for use in household water filters in rural areas of the developing world. However, improved medium preparation to further enhance its efficiency is needed; also, its efficacy in removing/inactivating indigenous enteric bacteria, viruses and protozoa has to be ensured and practicalities or economics of application need to be considered.

Effect of copper (II) on survival of Pseudomonas fluorescens and transfer of plasmid RP4 in soil

Survival of Pseudomonas fluorescens (R2f, RP4), P. fluorescens (R2f) and plasmid RP4 transfer efficiency depended on the concentration of Cu (II) and the time of incubation. The indigenous heterotrophic bacteria were more tiolerant to Cu (II) than the introduced strains. Although transconjugants were present for the first 3 days, the frequencies of conjugation and absence of transconjugants at 14 days indicate that conjugation occurred early in the incubation and that transconjugants were unable to survive for very long.