Bacterial toxicity comparison between nano- and micro-scaled oxide particles

Toxicity of nano-scaled aluminum, silicon, titanium and zinc oxides to bacteria (Bacillus subtilis, Escherichia coli and Pseudomonas fluorescens) was examined and compared to that of their respective bulk (micro-scaled) counterparts. All nanoparticles but titanium oxide showed higher toxicity (at 20 mg/L) than their bulk counterparts. Toxicity of released metal ions was differentiated from that of the oxide particles. ZnO was the most toxic among the three nanoparticles, causing 100% mortality to the three tested bacteria. Al₂O₃ nanoparticles had a mortality rate of 57% to B. subtilis, 36% to E. coli, and 70% to P. fuorescens. SiO₂ nanoparticles killed 40% of B. subtilis, 58% of E. coli, and 70% of P. fluorescens. TEM images showed attachment of nanoparticles to the bacteria, suggesting that the toxicity was affected by bacterial attachment. Bacterial responses to nanoparticles were different from their bulk counterparts; hence nanoparticle toxicity mechanisms need to be studied thoroughly.     

Effect of chlorination and ultraviolet disinfection on tetA-mediated tetracycline resistance of Escherichia coli

Antibiotic-resistant bacteria are an emerging threat to public health during drinking water consumption and reclaimed water reuse. Several studies have shown that the proportions of antibiotic-resistant bacteria in waters may increase when exposed to low doses of UV light or chlorine. In this study, inactivation of tetracycline-resistant Escherichia coli and antibiotic-sensitive E. coli by UV disinfection and chlorination was compared to determine the tolerance of tetracycline-resistant E. coli to UV light and chlorine, and tetracycline resistance of a tetracycline-resistant E. coli population was studied under different doses of the disinfectants. Our results showed that relative to antibiotic-sensitive E. coli, tetracycline-resistant E. coli had the same tolerance to UV light and a potentially higher tolerance to chlorination. The mortality frequency distributions of tetracycline-resistant E. coli exposed to tetracycline were shifted by both chlorination and UV disinfection. When compared to the hemi-inhibitory concentrations (IC₅₀) of tetracycline-resistant E. coli with no exposure to UV or chlorination, the IC₅₀ of tetracycline-resistant E. coli treated with tetracycline was 40% lower when inactivation by UV light or chlorination reached 3-log but was 1.18 times greater when inactivation by chlorination reached 4.3-log. Chlorination applied to drinking water or reclaimed water treatment may increase the risk of selection for highly tetracycline-resistant E. coli.     

水质对紫外消毒在两种典型再生水中应用的影响

以大肠杆菌为对象，研究了再生水水质变化对紫外消毒效果和光复活的影响。结果表明，紫外对大肠杆菌有很强的灭活作用，在紫外剂量为4mJ／cm^2时，大肠杆菌的灭活率达到了4．41个对数级。腐殖酸、铁和2种再生水水体中其他溶解性物质会影响光吸收和紫外透射率，但对紫外消毒动力学无影响。在颗粒物浓度为0～200mg／L的范围内，外源高岭土和活性污泥等颗粒物的投加对紫外消毒效果影响较小，而再生水水样W1中原有的2．61／1：g／L的颗粒物则会极大地影响消毒效果，使UV对细菌的灭活出现明显的拖尾现象。腐殖酸会增强紫外损伤大肠杆菌的光复活能力，但2种再生水中细菌的光复活能力相对磷酸盐缓冲溶液（PBS）中减弱，减弱程度在不同水样中有所不同。     

Occurrence of diarrheagenic Escherichia coli genes in raw water of water treatment plants

Purpose

The high incidences of waterborne diseases are frequently associated with diarrheagenic Escherichia coli (DEC). DEC may pose a health risk to people who contact surface water for recreation or domestic use. However, there is no published report on the monitoring of DEC in drinking water sources in Taiwan. In this study, the occurrence of DEC genes in raw water for water treatment plants in Taiwan was investigated.

Method

Raw water samples were taken from water treatment plants adjacent to the Kaoping River in southern Taiwan. Each water sample was treated with membrane filtration followed by DNA extraction from the concentrate and concentrate enrichment, respectively. The target genes for various DEC strains of genes were identified, including enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC).

Results

Among 55 water samples analyzed, DEC genes were detected in 16 (29.1%) samples. Strain-specific genes for EAEC, EHEC, EIEC, and EPEC were found in the percentages of 3.6%, 10.9%, 9.1%, and 9.1%, respectively. The specific gene for ETEC is not detected in the study. By looking at the presence/absence of specific genes and water sample characteristics, water temperature was found to differ significantly between samples with and without EHEC gene. In addition, pH levels differed significantly for EHEC and EPEC presence/absence genes, and turbidity was significantly different for water with and without EPEC genes.

Conclusion

DEC genes were detected in 29.1% of the raw water samples in the study location. The potential health threat may be increased if the treatment efficiencies are not properly maintained. Routine monitoring of DEC in drinking water sources should be considered.     

Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland)

This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.     

Variation of raw wastewater microbiological quality in dry and wet weather conditions

The microbiological quality of urban wastewaters presents important environmental, sanitary, and political challenges. However, the variability of untreated wastewater quality is seldom known when it comes to microbial parameters. This study aims to evaluate the variability of microbiological quality in wastewater influents from different wastewater treatment plants connected to combined and partially separate sewer networks in the Parisian area and to evaluate the impact of this variability on the treatment efficiency and on the accuracy of wastewater effluent monitoring. The densities of fecal indicator bacteria (FIB), Escherichia coli and intestinal enterococci, and their partitioning on settleable particles were analyzed at the inlet of two wastewater treatment plants during dry weather (130 composite samples and 7 days sampled every 2 hours) and storm events (39 composite samples, and 7 rain courses) from 2008 to 2012. The results showed that fecal indicator densities vary according to the network characteristics and according to the meteorological conditions. During storm events, a significant dilution of E. coli and enterococci was observed, as well as a decrease in the settleable fraction of E. coli during the maximal impact of the storm. However, storm events did not significantly impact the regular FIB monitoring. FIB removals by primary and secondary treatment were significantly correlated with FIB densities in influent wastewater; however, meteorological conditions also influenced the removal of FIB.     

Point-of-use water disinfection using UV light-emitting diodes to reduce bacterial contamination

The treatment process described in this research explores the impact of exposing water samples containing fecal coliforms to the radiation produced by single ultraviolet (UV) light-emitting diodes (LEDs) operating at 265 nm. UV LEDs are long lasting, compact in size and produce more efficient light output than traditional mercury-vapour bulbs, making them ideal for application in point-of-use disinfection systems, such as in remote areas. In this study, contaminated water samples containing either a pure culture of Escherichia coli or tertiary effluent from the City of Regina Wastewater Treatment Plant were used to study the application and efficiency of using UV LEDs for water disinfection. The results indicate that bacterial inactivation was achieved in a time-dependent manner, with 1- and 2.5-log E. coli reductions in water following 20 and 50 min of UV LED exposure, respectively. Ultraviolet radiation was less effective in reducing coliform bacteria in wastewater samples due to the elevated turbidity levels. Further work remains to be completed to optimize the application of UV LEDs for point-of-use disinfection systems; however, the results from this study support that bacterial inactivation using UV LEDs is possible, meriting further future technological development of the LEDs.     

Accumulated sediments in a detention basin: chemical and microbial hazard assessment linked to hydrological processes

Accumulated sediments in a 32,000-m³ detention basin linked to a separate stormwater system were characterized in order to infer their health hazards. A sampling scheme of 15 points was defined according to the hydrological behaviour of the basin. Physical parameters (particle size and volatile organic matter content) were in the range of those previously reported for stormwater sediments. Chemical analyses on hydrocarbons, PAHs, PCBs and heavy metals showed high pollutant concentrations. Microbiological analyses of these points highlighted the presence of faecal indicator bacteria (Escherichia coli and intestinal enterococci) and actinomycetes of the genus Nocardia. These are indicative of the presence of human pathogens. E. coli and enterococcal numbers in the sediments were higher at the proximity of the low-flow gutter receiving waters from the catchment. These bacteria appeared to persist over time among urban sediments. Samples highly contaminated by hydrocarbons were also shown to be heavily contaminated by these bacteria. These results demonstrated for the first time the presence of Nocardial actinomycetes in such an urban context with concentrations as high as 11,400 cfu g^?1.     

Faecal pollution loads in the wastewater effluents and receiving water bodies: a potential threat to the health of Sedibeng and Soshanguve communities,South Africa

The discharge of untreated or inadequately treated effluents has been identified among the activities responsible for the spread of a wide range of potentially infectious agents. The aim of this study was to determine whether inadequate treatment of wastewater and the faecal pollution load of effluents and receiving water bodies in Sedibeng District and Soshanguve peri-urban area of the Tshwane Metropolitan Municipality could be a potential threat to the health of the surrounding communities. Variations in the counts of faecal indicator bacteria and pathogenic microorganisms and compliance of the effluents and receiving water bodies with South African and World Health Organization standards were assessed between August 2011 and May 2012 using culture-based methods and molecular techniques. The overall quality of effluents did not comply with the South African special standard of no risk for unrestricted irrigation (zero?Escherichia coli/100 ml). The quality of the receiving water bodies did not comply with South African regulatory limits set for domestic purposes (zero?E.?coli/100 ml, <30 faecal enterococci/100 ml and <1 somatic coliphages/100 ml), for full contact recreation (<20 somatic coliphages/100 ml) and aquaculture (<10?E.?coli/100 ml) and WHO standards for full and intermediate contact recreational use (<1?E.?coli/100 ml and <40 faecal enterococci/100 ml, respectively). The PCR results revealed the prevalence of pathogenic microorganisms; between 0 and 60 % of samples tested positive for Salmonella Typhimurium and Shigella dysenteriae, and between 20 and 60 % of samples tested positive for Vibrio cholerae. These findings demonstrated that potential health risks might be associated with the use of the target river waters for domestic, recreational and irrigation purposes. This study calls for a prompt intervention to improve wastewater management.     

Visible light photocatalytic antibacterial activity of Ni-doped and N-doped TiO<Subscript>2</Subscript> on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> bacteria

The Ni-doped and N-doped TiO₂ nanoparticles were investigated for their antibacterial activities on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria. Their morphological features and characteristics such as particle size, surface area, and visible light absorbing capacity were compared and discussed. Scanning electron microscopy, X-ray diffraction, and UV–visible spectrophotometry were used to characterize both materials. The inactivation of E. coli (as an example of Gram-negative bacteria) and S. aureus (as an example of Gram-positive bacteria) with Ni-doped and N-doped TiO₂ was investigated in the absence and presence of visible light. Antibacterial activity tests were conducted using undoped, Ni-doped, and N-doped TiO₂. The N-doped TiO₂ nanoparticles show higher antibacterial activity than Ni-doped TiO₂. The band gap narrowing of N-doped TiO₂ can induce more visible light absorption and leads to the superb antibacterial properties of this material. The complete inactivation time for E. coli at an initial cell concentration of 2.7?×?10⁴ CFU/mL was 420 min which is longer than the 360 min required for S. aureus inactivation. The rate of inactivation of S. aureus using the doped TiO₂ nanoparticles in the presence of visible light is greater than that of E. coli. The median lethal dose (LD50) values of S. aureus and E. coli by antibacterial activity under an 18-W visible light intensity were 80 and 350 mg/ml for N-doped TiO₂, respectively.     

Alternative indicators for monitoring Cryptosporidium oocysts in reclaimed water

With the widespread use of reclaimed water all over the world, there is a clear need to optimise its management in order to guarantee water safety. Model microorganisms (with either indicator or index function) are commonly used to assess risks related to the presence of enteric pathogens in water. Samples from five water reclamation plants located in Northeastern Spain were analysed to validate the use of three model microorganisms (Escherichia coli, somatic coliphages and spores of sulphite-reducing clostridia) as surrogates of Cryptosporidium total or infectious oocysts (TOO and IOO, respectively) in reclaimed water. Probability plots, simple and multiple linear regression and discriminant analyses were performed to assess their relationships. Results show that the detection of E. coli alone is not useful to model either the behaviour or concentrations of Cryptosporidium. However, discriminant analyses showed a high rate of correctly classified samples (91.9 %) when E. coli and somatic coliphages data were used together to predict the presence/absence of IOO. Spores of sulphite-reducing clostridia (SRC) showed parallel reduction patterns and high correlation values (r?=?0.76) with reductions in TOO. Furthermore, simple regression analyses of SRC and TOO in reclaimed water showed high correlation values (r?=?0.85). Therefore, at the treatment plants studied, SRC can be considered to have good indicator and index functions for TOO. From the point of view of health protection, the use of SRC together with E. coli (which is mandatory in the current Spanish regulations) would satisfy the need for improved reclaimed water management.     

Tetracycline photolysis in natural waters: loss of antibacterial activity

Previous work has shown that tetracycline undergoes direct photolysis in the presence of sunlight, with the decomposition rate highly dependent on conditions such as water hardness and pH. The purpose of this study was to examine the potential long-term significance of photoproducts formed when tetracycline undergoes photodegradation under a range of environmentally relevant conditions. Tetracycline was photolyzed in nine different natural and artificial water samples using simulated sunlight. The pH values of the samples ranged from 5 to 9. Total hardness values (combined Ca²⁺ and Mg²⁺ concentrations) varied from 30 to 450 ppm. Assays based on growth inhibition of two bacterial strains, Escherichia coli DH5α and Vibrio fischeri, were used to determine the antibacterial activity of tetracycline’s photoproducts in these water samples. In all tested conditions, it was determined that the photoproducts retain no significant antibacterial activity; all observed growth inhibition was attributable to residual tetracycline. This suggests that tetracycline photoproducts formed under a wide range of pH and water hardness conditions will not contribute to the selection of antibiotic-resistant bacteria in environmental systems.     

Dechlorination and organohalide-respiring bacteria dynamics in sediment samples of the Yangtze Three Gorges Reservoir

Several groups of bacteria such as Dehalococcoides spp., Dehalobacter spp., Desulfomonile spp., Desulfuromonas spp., or Desulfitobacterium spp. are able to dehalogenate chlorinated pollutants such as chloroethenes, chlorobenzenes, or polychlorinated biphenyls under anaerobic conditions. In order to assess the dechlorination potential in Yangtze sediment samples, the presence and activity of the reductively dechlorinating bacteria were studied in anaerobic batch tests. Eighteen sediment samples were taken in the Three Gorges Reservoir catchment area of the Yangtze River, including the tributaries Jialing River, Daning River, and Xiangxi River. Polymerase chain reaction analysis indicated the presence of dechlorinating bacteria in most samples, with varying dechlorinating microbial community compositions at different sampling locations. Subsequently, anaerobic reductive dechlorination of tetrachloroethene (PCE) was tested after the addition of electron donors. Most cultures dechlorinated PCE completely to ethene via cis-dichloroethene (cis-DCE) or trans-dichloroethene. Dehalogenating activity corresponded to increasing numbers of Dehalobacter spp., Desulfomonile spp., Desulfitobacterium spp., or Dehalococcoides spp. If no bacteria of the genus Dehalococcoides spp. were present in the sediment, reductive dechlorination stopped at cis-DCE. Our results demonstrate the presence of viable dechlorinating bacteria in Yangtze samples, indicating their relevance for pollutant turnover.     

Fecal contamination in irrigation water and microbial quality of vegetable primary production in urban farms of Metro Manila,Philippines

Microbial contamination of fresh produce can present a severe risk to public health. By conducting a rigorous survey of irrigation waters, the impacts of fecal contamination on the quality of produce could be assessed. In this study, surface waters were observed to be contaminated with Escherichia coli, Salmonella spp., and somatic coliphages. Culture methods show that out of 373 irrigation water, soil, and vegetable samples collected for a 1-year period, 232 (62.20%) were found positive for E. coli, 213 (57.26%) for somatic coliphages, and 2 (0.53%) for Salmonella spp. Out of 190 water samples, 167 (87.9%) were found to have E.coli, 174 (91.6%) have somatic coliphages, and 1 (0.5%) with Salmonella spp. In soil samples, 36 of 91 (39.6%) have E. coli, 31 (34.0%) have somatic coliphages, and none with Salmonella spp. Lastly, out of 92 vegetable samples, 29 (31.5%), 8 (8.7%), and 1 (1.1%) were found to have E. coli, somatic coliphages, and Salmonella spp., respectively. Molecular analysis confirmed the presence of bacterial contaminants. Seasonal weather conditions were noted to have an effect on the presence and number of these fecal indicator organisms. The observed data suggest that contaminated irrigation water may greatly affect the quality of fresh produce from these agricultural operations.     

Microbial and physicochemical parameters associated with Legionella contamination in hot water recirculation systems

Hot water recirculation systems (HWRS) in hotels and nursing homes, which are common in countries such as Spain, have been related to outbreaks of legionellosis. To establish the relationships of microbial and physicochemical parameters, especially protozoa, with the occurrence of Legionella in HWRS, 231 samples from hotels and nursing homes were analysed for Legionella, protozoa, heterotrophic plate counts (HPC) at 22 and 37 °C, Pseudomonas, metals, temperature and others. Legionella pneumophila was the dominant species isolated, and 22 % were sg. 1. The sampling method became particularly important in order to define which factors were involved on the occurrence of Legionella. Results showed that the bacteria and the accompanying microbiota were more abundant in the first flush water whose temperature was lower. The bacteria occurred in those samples with high HPC and were inversely correlated with high temperatures. Multivariate regression showed that a concentration above 1?×?10⁵?CFU/100 mL of HPC at 37 °C, Fe above 0.095 ppm and the presence of protozoa increased significantly the risk of Legionella colonization, while univariant regression showed that the presence of Cu above 0.76 ppm and temperature above 55 °C diminished it. Therefore, to reduce the risk associated with Legionella occurrence in HWRS these parameters should be taken into consideration.     

In vivo exposure of Mytilus edulis to living enteric bacteria: a threat for immune competency?

Mussels are widespread in coastal environments and experience various physical, chemical, and bacteriological conditions. Owing to the increase of coastal urbanization, mussels are now commonly exposed not only to indigenous bacteria, but also to enteric bacteria originating from pulsed and chronic sewage discharges into coastal environments. Due to its broad resilience to environmental variations, the blue mussel Mytilus edulis is commonly used as an indicator of environmental quality in bio-monitoring programs. However, since mussel immune system capabilities may be affected by the presence of exogenous fecal bacteria in coastal seawater subjected to sewage discharges, we aimed to determine the effect of in vivo bacterial challenges on mussels' immune competency by using two exogenous enteric bacterial strains, Escherichia coli and Enterococcus faecalis, and an indigenous bacterial strain Vibrio splendidus (as control). Bacterial strains were tested individually, by injection into the posterior adductor muscle at three different cell densities (10², 10³, and 10⁴ cells). Unlike classic in vitro experiments using higher bacterial concentrations, neither the enteric bacteria nor the indigenous strain induced significant increase or decrease of either cell-mediated (phagocytosis, reactive oxygen species, and NO _x production) or humoral components (prophenoloxidase-like, acid phosphatase, and l-leucine-aminopeptidase production) of the immune system. This study demonstrates that, at low concentrations, E. coli and E. faecalis do not represent an additional threat that could impair M. edulis immune competency and, as a consequence, its potential of survival in coastal areas subjected to sewage discharges.     

Assessment of cytotoxicity and toxicity for phosphonium-based deep eutectic solvents

In this work, the cytotoxicity and toxicity of phosphonium-based deep eutectic solvents (DESs) with three hydrogen bond donors, namely glycerine, ethylene glycol, and triethylene glycol were investigated. The cytotoxicity effect was tested using brine shrimp (Artemia salina). The toxicity was investigated using the two Gram positive bacteria Bacillus subtilis and Staphylococcus aureus, and two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The cytotoxicity of tested DESs was much higher than that of their individual components, indicating their toxicological behavior was different. It was also found that there was toxic effect on the studied bacteria, indicating their potential application as anti-bacterial agents. To the best of our knowledge, this is the first time the cytotoxicity and toxicity of phosphonium-based DESs were studied.     

Monitoring of genetically modified Escherichia coli in laboratory wastewater

Containment of genetically modified (GM) microorganisms such as Escherichia coli is a legal requirement to protect the environment from an unintended release and to avoid horizontal gene transfer (HGT) of recombinant DNA to native bacteria. In this study, we sampled the laboratory wastewater (LWW) at a large Swiss university from three sources over 2 years and cultured ampicillin-resistant, presumptive GM E. coli. From a total of 285 samples, 127 contained presumptive GM E. coli (45%) at a mean concentration of 2.8 × 10² CFU/ml. Plasmid DNA of 11 unique clones was partially or entirely sequenced. All consisted of cloning vectors harboring research-specific inserts. To estimate the chance of HGT between GM E. coli and native bacteria in LWW, we identified taxa representative for the bacterial community in LWW using 16S rRNA amplicon sequencing and measured conjugation frequencies of E. coli with five LWW isolates. At optimal conjugation conditions, frequencies were between 3.4 × 10⁻³ and 2.4 × 10⁻⁵. Given the absence of transferable broad-host range plasmids and suboptimal conjugation conditions in the LWW system, we conclude that the chance of HGT is relatively low. Still, this study shows that the implementation of robust containment measures is key to avoid the escape of GM microorganisms.

     

Evaluation and use of a biotracer to study ground water contamination by leaching bed systems

There is an increasing concern about dangerous levels of bacterial contamination of rural ground water resources in Ontario and throughout the world. Recent studies in rural parts of Canada have identified leaching bed systems as one of the major sources of this contamination. Field studies were undertaken to evaluate bacterial contamination from three different types of leaching bed designs, using nalidixic acid-resistant Escherichia coli (E. coli NAR) as a biotracer. This biotracer was used rather than passive ground water sampling to clearly identify the source of the contamination and also to allow the determination of travel times and distances more clearly. While this biotracer has been used for other studies its use in actual working septic systems has not yet been reported.This work has also shown that E. coli NAR is an excellent biotracer and can be used to give an accurate assessment of a leaching bed's performance provided it is introduced into the system over a reasonable period of time. Results also show that bacteria are not necessarily removed before the effluent reaches the ground water. The speed, distance of travel and attenuation of biotracer concentrations was found to be highly related to precipitation events, age of system and depth of unsaturated zone below the bed.     

Development of eco-friendly bioplastic like PHB by distillery effluent microorganisms

During screening for poly-β-hydroxybutyrate (PHB) producing bacteria from distillery effluent sample, six out of 30 isolates comprising of three strains of Alcaligenes sp., two strains of Bacillus sp., and one strain of Pseudomonas sp. were found to accumulate varying levels of intracellular PHB. Amongst the various isolates, Alcaligenes sp. RZS4 was found as the potent PHB-producing organism, accumulating higher amounts of PHB. PHB productivity was further enhanced in the presence of oxygen, nitrogen-limiting conditions, and cloning of PHB synthesizing genes of Alcaligenes sp. RZS 4 into Escherichia coli. A twofold increase in PHB yield was obtained from recombinant E. coli vis-à-vis Alcaligenes sp.; the recombinant E. coli accumulated more PHB in NDMM, produced good amount of PHB in a single-stage cultivation process under both nutrient-rich and nutrient-deficient conditions. Extraction of PHB with acetone–alcohol (1:1) was found as suitable method for optimum extraction of PHB as this mixture selectively extracted PHB without affecting the non-PHB cell mass. PHB extract from recombinant E. coli showed the presence of C–H, =O stretching, =C–H deformation, =C–H, =CH, and =C–O functional groups characteristic of PHB.