Interactions between heavy metals and bacteria in mangroves.

Environmental heavy metal pollution has become a serious problem in recent years. Therefore, our study investigated seven heavy metal-contaminated mangroves (Beihai, Fangchenggang, Hainan, Hongkong, Shenzhen, Yunxiao, and Zhanjiang) in southern China, and found that they were particularly polluted with Zn and Pb. These heavy metals were mainly distributed in the surface sediments of the mangroves. Among these seven mangroves, the Shenzhen mangrove was the most polluted site, whereas the Beihai mangrove was the least polluted. Moreover, the bacterial communities in the mangroves were significantly associated with heavy metal contamination. For instance, Fusibacter was significantly correlated with Pb, Zn, Cu, Co, Ni, Cd, and Ag (P < 0.05, R = -0.47). Syntrophorhabdus was also significantly correlated with heavy metals (P < 0.05, R = 0.63). Furthermore, Geo-Chip analyses were conducted to demonstrate the involvement of many functional genes in heavy metal transport, particularly Ni transport. Our results also demonstrated that the heavy metals could be transported by various bacteria. For example, Pseudomonas and Burkholderia were involved in various heavy metal transportation mechanisms, particularly for Ni and Zn, suggesting that these bacteria could be used for heavy metal remediation. Therefore, our study provides insights into the interactions between bacterial communities and heavy metals, which could enable the development of novel mangrove protection strategies.

Biotransformation strategies for steroid estrogen and androgen pollution.

The common steroid hormones are estrone (E1), 17ß-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), and testosterone (T). These steroids are reported to contaminate the environment through wastewater treatment plants. Steroid estrogens are widespread in the aquatic environment and therefore pose a potential risk, as exposure to these compounds has adverse impacts on vertebrates. Excessive exposure to steroid estrogens causes endocrine disruption in aquatic vertebrates, which affects the normal sexual life of these animals. Steroid pollutants also cause several health problems in humans and other animals. Microbial degradation is an efficient method for removing hormone pollutants from the environment by remediation. Over the last two decades, microbial metabolism of steroids has gained considerable attention due to its higher efficiency to reduce pollutants from the environment. The present review is focused on the major causes of steroid pollution, concentrations of these pollutants in surface water, groundwater, drinking water, and wastewater, their effect on humans and aquatic animals, as well as recent efforts by various research groups that seek better ways to degrade steroids by aerobic and anaerobic microbial systems. Detailed overview of aerobic and anaerobic microbial biotransformation of steroid estrogens and testosterone present in the environment along with the active enzyme systems involved in these biotransformation reactions is described in the review article, which helps readers to understand the biotransformation mechanism of steroids in depth. Other measures such as co-metabolic degradation, consortia degradation, algal, and fungal steroid biotransformation are also discussed in detail.

Identification of non-accumulating intermediate compounds during estrone (E1) metabolism by a newly isolated microbial strain BH2-1 from mangrove sediments of the South China Sea.

Steroid estrogens are natural hormonal compounds produced by various animals and humans. Estrone (E1), estradiol (E2), and estriol (E3) are the most commonly known estrogens that are released into the environment along with human and animal excreta, which end up polluting water bodies. While these estrogens are usually biotransformed into their respective by-products by various microbial strains, E2 could also be transformed into E1 by 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) under reducing environmental conditions. However, due to limited further biotransformation of E1, it accumulates to higher levels in water bodies compared to other natural estrogens in the aquatic environment. Given that E1 is one of the potential endocrine-disrupting compounds (EDCs), with several adverse effects on aquatic animals and consequently on the seafood industry, it is vital to remove E1 from the environment via improved steroid bioremediation. In the present study, we successfully isolated a potential E1-degrading microbial strain (named as BH2-1) from soil sediments collected from the Bai Hai mangrove region of the South China Sea. The strain BH2-1 has excellent E1-degrading potential and could degrade 89.5% of E1 after 6 days of incubation in a MSM-E1 medium containing 1% NaCl at pH 6. Besides, after 3 h and 6 h of extraction, two non-accumulating intermediate compounds [3-hydroxyandrosta-5,7,9(11)-trien-17-one and androsta-1,4,6-triene-3,17-dione (ATD)], respectively, were successfully identified using GC-MS analysis. These non-accumulating intermediate compounds have not previously been reported during E1 biodegradation and might be new intermediate metabolites. The identification of these new compounds also gives more insight into the mechanism of E1 metabolism and helps to establish a clear E1 biodegradation pathway, which further enriches our knowledge on the overall microbial steroid degradation pathway. Furthermore, whole-genome sequence analysis of strain BH2-1 revealed the presence of 46 genes that belong to 6 major steroid-degrading gene classes.

Aislamiento y caracterización de una bacteria marina degradadora de testosterona: Vibrio sp. N3 / Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3

Steroids, including testosterone, estrone, 17β-estradiol, estriol and 17β-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17β-estradiol (E2), 17β-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48 h.

Los esferoides-que incluyen la testosterona, la estrona, el 17 β-estradiol, el estriol y el 17 p-etinilestradiol-son nocivos no solo para la población dinámica de las formas de vida acuática, sino también para la salud pública. En este estudio se aisló una bacteria marina degradadora de testosterona de la isla de Nanao, en el Mar del Sur de China, a la que se denominó cepa N3. Se determinó que esta cepa también podría usar 17 β-estradiol (E2), 17 p-etinilestradiol (EE2), estriol (E3) o colesterol como únicas fuentes de carbono. De acuerdo con el análisis de la secuencia del gen 16S rRNA, la cepa N3 se identificó como Vibrio sp. La caracterización adicional mostró que dicha bacteria es un organismo aerobio, gram negativo y móvil, y que presenta resistencia a ampicilina, carbenicilina, penicilina y espectinomicina. Para optimizar la condición de cultivo en relación con su capacidad de degradar la testosterona, se utilizaron el diseño factorial Plackett-Burman y el diseno compuesto central. En condiciones óptimas, el 92% de la testosterona fue degradada por Vibrio sp. N3 en 48 h.

Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3.

Steroids, including testosterone, estrone, 17ß-estradiol, estriol and 17ß-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17ß-estradiol (E2), 17ß-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48h.

A novel dehydrogenase 17ß-HSDx from Rhodococcus sp. P14 with potential application in bioremediation of steroids contaminated environment.

Steroids are endocrine disrupting compounds in human and are distributed in various environments. Our previous study showed that a marine bacterium Rhodococcus sp. P14 was able to efficiently degrade one typical steroid estradiol. In this study, we showed that P14 could also use other steroids, including estriol and testosterone, as sole carbon source for growth. Two dehydrogenation products, 16-hydroxestrone and androst-4-ene-3, 17-dione, were detected during estriol and testosterone degradation, respectively. By screening the genome, a short chain dehydrogenase gene was identified and named as 17ß-HSDx. Expression of 17ß-HSDx was induced in P14 when estriol, estradiol or testosterone was used as single carbon source. In addition, 17ß-HSDx was shown to have dehydrogenation ability of transforming estriol to 16-hydroxestrone, estradiol to estrone and testosterone to androst-4-ene-3, 17-dione. This is the first short chain dehydrogenase identified in bacteria with dehydrogenation ability on various steroids substrates. Overall, this study reveals that 17ß-HSDx has potential application in the bioremediation of steroids contaminated environment.

Acclimation of Culturable Bacterial Communities under the Stresses of Different Organic Compounds.

The phylogenetic diversity of bacterial communities in response to environmental disturbances such as organic pollution has been well studied, but little is known about the way in which organic contaminants influence the acclimation of functional bacteria. In the present study, tolerance assays for bacterial communities from the sediment in the Pearl River Estuary were conducted with the isolation of functional bacteria using pyrene and different estrogens as environmental stressors. Molecular ecological networks and phylogenetic trees were constructed using both 16S rRNA gene sequences of cultured bacterial strains and 16S rRNA gene-based pyrosequencing data to illustrate the successions of bacterial communities and their acclimations to the different organic compounds. A total of 111 bacterial strains exhibiting degradation and endurance capabilities in response to the pyrene estrogen-induced stress were successfully isolated and were mainly affiliated with three orders, Pseudomonadales, Vibrionales, and Rhodobacterales. Molecular ecological networks and phylogenetic trees showed various adaptive abilities of bacteria to the different organic compounds. For instance, some bacterial OTUs could be found only in particular organic compound-treated groups while some other OTUs could tolerate stresses from different organic compounds. Furthermore, the results indicated that some new phylotypes were emerged under stresses of different organic pollutions and these new phylotypes could adapt to the contaminated environments and contribute significantly to the microbial community shifts. Overall, this study demonstrated a crucial role of the community succession and the acclimation of functional bacteria in the adaptive responses to various environmental disturbances.

Adverse effect of heavy metals (As, Pb, Hg, and Cr) on health and their bioremediation strategies: a review.

Currently, heavy metal pollution becomes a severe problem whole over the world, and these toxic metals enter into the environment either by natural phenomena or due to extensive industrialization. The discharged effluents containing toxic heavy metals mixed with soil/water and change their natural composition. These heavy metals have adverse effects on living beings and cause damage to the vital body organs of animals as well as humans. The heavy metal pollution also inhibits the biodegradation of the chlorinated organic compounds (another type of environmental pollution) by interacting with metabolizing enzymes and inhibits their functioning. Earlier studies described that heavy metals cannot be fully removed from the environment, but they can be effectively neutralized or transformed into less toxic form so that their harmful effect on the environment can be reduced. The distinctive enzymatic apparatus within microbial system plays a major role in the transformation of heavy metals in the environment. A considerable advancement has been made during recent years to transform the heavy metals by utilizing the bioremediation potential of genetically engineered (GE) microorganisms. These transgenics are very much efficient in heavy metal transformations and still, we have to discover more to additionally utilize their full biotransformation potential.In the present review article, the detailed description of the adverse effects of four heavy metals (arsenic, lead, mercury, and chromium) and their adverse effect on our environment and human beings is discussed. Furthermore, the use of microorganisms/GE organisms for the bioremediation of heavy metals from the environment is also discussed along with their detailed bioremediation mechanism.

An innovative ring-shaped electroeluter for high concentration preparative isolation of protein from polyacrylamide gel.

A ring-shaped electroeluter (RSE) was designed for protein recovery from polyacrylamide gel matrix. The RSE was designed in such a way that a ring-shaped well was used to place gel slices and an enrichment well was used to collect eluted protein samples. With HSA as model protein, the electroelution time was less than 30 min with 80% recovery rate, and the concentration of recovered protein was 50 times higher than that of conventional method. The RSE could be reused at least ten times. The developed device makes great advance towards economic electroelution of biomolecules (such as proteins) from gel matrix.

In-Vial Temperature Gradient Headspace Single Drop Microextraction Designed by Multiphysics Simulation.

Presented herein is a novel headspace single drop microextraction (HS-SDME) based on temperature gradient (TG) for an on-site preconcentration technique of volatile and semivolatile samples. First, an inner vial cap was designed as a cooling device for acceptor droplet in HS-SDME unit to achieve fast and efficient microextraction. Second, for the first time, an in-vial TG was generated between the donor phase in a sample vial at 80 °C and the acceptor droplet under the inner vial cap containing cooling liquid at -20 °C for a TG-HS-SDME. Third, a simple mathematic model and numerical simulations were developed by using heat transfer in fluids, Navier-Stokes and mass balance equations for conditional optimization, and dynamic illumination of the proposed extraction based on COMSOL Multiphysics. Five chlorophenols (CPs) were selected as model analytes to authenticate the proposed method. The comparisons revealed that the simulative results were in good agreement with the quantitative experiments, verifying the design of TG-HS-SDME via the numerical simulation. Under the optimum conditions, the extraction enrichments were improved from 302- to 388-fold within 2 min only, providing 3.5 to 4 times higher enrichment factors as compared to a typical HS-SDME. The simulation indicated that these improvements in the extraction kinetics could be attributed due to the applied temperature gap between the sample matrix and acceptor droplet within the small volume of headspace. Additionally, the experiments demonstrated a good linearity (0.03-100 µg/L, R2 > 0.9986), low limit of detection (7-10 ng/L), and fair repeatability (<5.9% RSD, n = 6). All of the simulative and experimental results indicated the robustness, precision, and usefulness of TG-HS-SDME for trace analyses of analytes in a wide variety of environmental, pharmaceutical, food safety, and forensic samples.

Molecular diversity and functional variability of environmental isolates of Bacillus species.

In the present study, out of 264 phosphate (P) solubilizing Bacillus strains isolated from apple rhizosphere, only twelve isolates were found to be efficient (showed most of the plant growth promoting activity) which were further characterized at molecular level using 16S rDNA partial gene sequencing. Out of 12 isolates, MZPSB 207 was found to be most efficient P-solubilizing (864.71 µg/ml) isolate which also showed indole acetic acid production (51.83 µg/ml), siderophore production, ammonia production, antagonistic property (against Rhizoctonia solani and Fusarium oxysporum), hydrolytic enzymes productions (protease, chitinase and cellulase), 1-aminocyclopropane-1-carboxylate (ACC) deaminase production (7.7 µm αKB mg(-1) h(-1)). The in-vitro seed germination assay showed that Bacillus (twelve isolates) inoculated seeds showed more seed germination and seedling vigor rate as compared to uninoculated control treatment. For the genetic diversity studies of efficient 12 strains, the polyphasic approach using 16S-rDNA, Repetitive element sequence (rep) based PCR (ERIC-PCR and BOX-PCR) were used. Based on 16S rDNA partial gene sequencing the isolated Bacillus genus was divide into four groups. First group (five isolates), second group (two isolates), third group (three isolates) and fourth group (two isolates) which showed close genetic relatedness to the B. subtilis, B. pumulis, B. megaterium and B. amyloliquefaciens, respectively. The rep PCR fingerprinting showed variability between and within the species. The large variability was showed by ERIC-PCR whereas some variability was showed by BOX-PCR. The results clearly showed that 16S rRNA gene sequencing is unable to discriminate the isolates at strain level. But rep-PCR fingerprinting is excellent tool to characterize and discriminate the strains at the genomic level.

Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34.

Nitrile hydratase (NHase; E.C. 4.2.1.84) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and ß-subunits with a molecular mass of 25 and 30 kDa, respectively. The Km and Vmax values were 102 mM and 350.8 µmol/min/mg using 3-cyanopyridine as substrate. The purified NHase was stable in higher concentration of potassium ions and in acidic pH 5.5 as compared to NHase of the wild R. rhodochrous PA-34. The analysis of the N-terminal amino acid sequence of this enzyme revealed that this enzyme has 90 % homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.

Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli.

The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria-Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

Generation of mutant of Rhodococcus rhodochrous PA-34 through chemical mutagenesis for hyperproduction of nitrile hydratase.

Rhodococcus rhodochrous PA-34 has been reported to produce nitrile hydratase enzyme that converts 3-cyanopyridine to nicotinamide. A mutant of R. rhodochrous PA-34 was generated through chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine (MNNG) that exhibited 2 times higher nitrile hydratase activity as compared to wild strain. The reaction conditions using resting cells of this mutant strain for the conversion of nicotinamide were optimized. Under the optimized reaction conditions the mutant strain exhibited maximum nitrile hydratase activity [7.8 U/mgdcm (milligram dry cell mass)] at 55 degrees C in 0.3 M potassium phosphate buffer (pH 5.5).