Inoculation of Bacillus megaterium strain A14 alleviates cadmium accumulation in peanut: effects and underlying mechanisms.

AIMS: A cadmium (Cd)-tolerant Bacillus megaterium strain A14 was used to investigate the effects and mechanisms of bacterial inoculation on peanut growth, Cd accumulation in grains and Cd fixation in Cd-contaminated soil. METHODS AND RESULTS: Spectroscopic analysis showed that A14 has many functional groups (-OH, -NH2 and -COO et al.) distributed on its surface. The pot experiment indicated that compared to the Cd-contaminated soil alone treatment, inoculation with strain A14 increased shoot and root biomass by 59·93 and 58·31% respectively. The accumulation of Cd in grains decreased by 48·14%, while the proportion of exchangeable Cd in soil decreased from 40 to 26% in A14 inoculated soil. CONCLUSIONS: Inoculation with B. megaterium A14 improved peanut plant growth via (i) adsorbing Cd2+ through functional groups on cell surface, (ii) immobilization of Cd in soil through extracellular secretions, (iii) scavenging the reactive oxygen species through production of antioxidant enzymes, and (iv) by reducing the phytoavailable Cd through regulation of Cd transport gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a new sight on microbial approach for the chemical composition transformation of soil Cd and associated food safety production, which pointed out an efficient way to improve peanut cultivation.

Parabens generate reactive oxygen species in human spermatozoa.

Parabens are used as antimicrobial preservative agent in many commercial products including cosmetics and pharmaceuticals. Weak oestrogenic and antiandrogenic activities have been attributed to parabens in in vitro and in vivo studies. In this study, human spermatozoa were exposed to different concentrations of an equimolar paraben mixture containing methyl, ethyl, propyl and butylparaben as well as to methylparaben alone at a concentration that is typical of commercially available vaginal lubricants. The induction of oxidative stress and DNA damage was then assessed at different time points. Our results demonstrate that the paraben mixture was capable of stimulating the generation of mitochondrial and cytosolic reactive oxygen species (ROS), inhibiting sperm motility and viability in a dose-dependent manner. The ability of individual parabens to activate ROS generation and induce oxidative DNA damage was related to alkyl chain length. At the concentration used clinically, methylparaben inhibited sperm motility after both 2 and 5 h exposure (p < 0.05) and affected cell viability (p < 0.01) while augmenting ROS production and oxidative DNA damage. However, DNA fragmentation was not evident following methylparaben exposure. Based on these results, we conclude that, at the concentrations used in commercially available formulations, parabens may impair sperm motility, enhance the generation of mitochondrial ROS and stimulate the formation of oxidative DNA adducts. Taken together, these data underline the potential cytotoxic and genotoxic impact of such compounds in a clinical setting.

The influence of biochar and black carbon on reduction and bioavailability of chromate in soils.

The widespread use of chromium (Cr) has a deleterious impact on the environment. A number of pathways, both biotic and abiotic in character, determine the fate and speciation of Cr in soils. Chromium exists in two predominant species in the environment: trivalent [(Cr(III)] and hexavalent [Cr(VI)]. Of these two forms, Cr(III) is nontoxic and is strongly bound to soil particles, whereas Cr(VI) is more toxic and soluble and readily leaches into groundwater. The toxicity of Cr(VI) can be mitigated by reducing it to Cr(III) species. The objective of this study was to examine the effect of organic carbon sources on the reduction, microbial respiration, and phytoavailability of Cr(VI) in soils. Organic carbon sources, such as black carbon (BC) and biochar, were tested for their potential in reducing Cr(VI) in acidic and alkaline contaminated soils. An alkaline soil was selected to monitor the phytotoxicity of Cr(VI) in sunflower plant. Our results showed that using BC resulted in greater reduction of Cr(VI) in soils compared with biochar. This is attributed to the differences in dissolved organic carbon and functional groups that provide electrons for the reduction of Cr(VI). When increasing levels of Cr were added to soils, both microbial respiration and plant growth decreased. The application of BC was more effective than biochar in increasing the microbial population and in mitigating the phytotoxicity of Cr(VI). The net benefit of BC emerged as an increase in plant biomass and a decrease in Cr concentration in plant tissue. Consequently, it was concluded that BC is a potential reducing amendment in mitigating Cr(VI) toxicity in soil and plants.

Atrazine and simazine degradation in Pennisetum rhizosphere.

The ability of rhizosphere of four plant species to promote the degradation of charcoal-fixed atrazine and simazine in cement blocks of a long-term contaminated soil when mixed with a normal soil at 1:1 ratio was tested. Of the four selected plants viz., rye grass (Lolium perenne), tall fescue (Festuca arundinacae), Pennisetum (Pennisetum clandestinum) and a spring onion (Allium sp.) used in this study, only P. clandestinum was able to survive in herbicide contaminated soil while other plants died within few days after germination/transplanting. Both atrazine and simazine were degraded at a faster rate in contaminated soil planted to P. clandestinum than in unplanted soil. Within 80 days, nearly 45% and 52% of atrazine and simazine, respectively, were degraded in soil planted to P. clandestinum while only 22% and 20% of the respective herbicide were degraded in the unplanted soil. During 80-day experimental period, both microbial biomass and soil dehydrogenase activity were significantly increased (7-fold) in soil planted to P. clandestinum over that in unplanted soil. The suspension of contaminated rhizosphere soil, planted to P. clandestinum exhibited an exceptional capability to degrade both atrazine (300 microg) and simazine (50 microg) in a mineral salts medium over that of non-rhizosphere soil suspension. Results indicate that P. clandestinum, a C4 plant, may be useful for remediation of soils contaminated with atrazine and simazine.

Algal degradation of a known endocrine disrupting insecticide, alpha-endosulfan, and its metabolite, endosulfan sulfate, in liquid medium and soil.

The role of algae in the persistence, transformation, and bioremediation of two endocrine disrupting chemicals, alpha-endosulfan (a cyclodiene insecticide) and its oxidation product endosulfan sulfate, in soil (incubated under light or in darkness) and a liquid medium was examined. Incubation of soil under light dramatically decreased the persistence of alpha-endosulfan and enhanced its transformation to endosulfan sulfate, over that of dark-incubated soil samples, under both nonflooded and flooded conditions. This enhanced degradation of soil-applied alpha-endosulfan was associated with profuse growth of indigenous phototrophic organisms such as algae in soil incubated under light. Inoculation of soil with green algae, Chlorococcum sp. or Scenedesmus sp., further enhanced the degradation of alpha-endosulfan. The role of algae in alpha-endosulfan degradation was convincingly demonstrated when these algae degraded alpha-endosulfan to endosulfan sulfate, the major metabolite, and endosulfan ether, a minor metabolite, in a defined liquid medium. When a high density of the algal inoculum was used, both metabolites appeared to undergo further degradation as evident from their accumulation only in small amounts and the appearance of an endosulfan-derived aldehyde. Interestingly, beta-endosulfan was detected during degradation of alpha-endosulfan by high density algal cultures. These algae were also capable of degrading endosulfan sulfate but to a lesser extent than alpha-endosulfan. Evidence suggested that both alpha-endosulfan and endosulfan sulfate were immediately sorbed by the algae from the medium, which then effected their degradation. Biosorption, coupled with their biotransformation ability, especially at a high inoculum density, makes algae effective candidates for remediation of alpha-endosulfan-polluted environments.

Microbial activity and phospholipid fatty acid pattern in long-term tannery waste-contaminated soil.

We investigated the phospholipid fatty acid (PLFA) pattern and dehydrogenase activity (DHA) in soil samples from three sites (designated as low, medium, and high based on the level of chromium) in a long-term (25 years after last waste input) tannery waste-contaminated area rich in Cr. Soil samples, collected from different soil depths (0-100 cm), at each site were used in this study. In general, soil samples from all three contaminated sites had elevated pH, electrical conductivity, organic carbon (OC), total Cr, and hexavalent Cr [Cr(VI)]. The maximum total Cr concentration in surface soils (0-10 cm) at the highly contaminated site was 102 gkg(-1), with 4.6 mgkg(-1) present as the bioavailable water-soluble Cr. More than 50% of soluble Cr was in the form of Cr(VI) (2.7 mgkg(-1)). DHA (normalized to OC) was inhibited to a greater extent in soil samples from the highly contaminated site than in low- and medium-contaminated soil samples. PLFA analyses of surface soils indicated that there was a shift in PLFA patterns. PLFAs specific for bacteria (i15:0, a15:0, 15:0, i16:0, a17:0, and cy17:0) decreased significantly (P<0.01) with an increase in Cr contamination. Among the bacterial PLFAs, 15:0, i16:0 and a17:0 had a significant negative correlation with contamination including bioavailable Cr(VI) in soil solution. To our knowledge, this is the first report of alterations in the PLFA profile in soils due to long-term tannery waste pollution.

Chemistry of chromium in soils with emphasis on tannery waste sites.

Worldwide chromium contamination of soils has arisen predominantly from the common practice of land-based disposal of tannery wastes under the assumption that the dominant species in the tannery waste would be the thermodynamically stable Cr(III) species. However, significant levels of toxic Cr(VI) recently detected in surface water and groundwater in India, China, Australia, and elsewhere raise critical questions relating to current disposal criteria for Cr-containing wastes. It now appears that despite the thermodynamic stability of Cr(III), the presence of certain naturally occurring minerals, especially Mn oxides, can enhance oxidation of Cr(III) to Cr(VI) in the soil environment. This factor is of public concern because at high pH, Cr(VI) is bioavailable, and it is this form that is highly mobile and therefore poses the greatest risk of groundwater contamination. A review of the current literature indicates that extensive research has been performed on the speciation of Cr in soil, the effect of pH on soil solution concentrations of Cr(III) and Cr(VI), soil adsorption phenomenon of Cr species, redox reactions, and transformation of Cr(II) and Cr(VI) together with remediation strategies to decontaminate Cr-contaminated soils. Most of the studies were conducted using an uncontaminated soil artificially spiked with Cr, and very limited research has been conducted in the contaminated soil environment. Furthermore, studies on tannery waste contaminated soils are limited, and obviously a serious gap of knowledge exists in understanding the influence of long-term tannery waste contamination on Cr behavior in soil.

Microbial degradation of illicit drugs, their precursors, and manufacturing by-products: implications for clandestine drug laboratory investigation and environmental assessment.

Chemicals associated with clandestine drug laboratories are often disposed of covertly into soil, sewerage systems, or public waste management facilities. There are two significant issues relating to such dumps of materials; they might contain valuable evidence as to drug manufacture, and they might be a source of pollution. This study presents initial findings in relation to the impact microorganisms from environmental sources have upon drugs, their precursors, and manufacturing by-products. The aim of this study was to identify which chemicals associated with clandestine drug laboratories persist in the environment in order to allow forensic drug chemists to link discarded residues with the method of manufacture, and to allow the environmental impact of clandestine drug laboratories to be assessed accurately. When exposed to soil microorganisms, phenyl-2-propanone (P2P) was rapidly metabolized into mixtures of 1-phenyl-2-propanol, 1-phenyl-1,2-propanedione, 1-hydroxy-1-phenyl-2-propanone, 2-hydroxy-1-phenyl-1-propanone, and the two diastereoisomers of 1-phenyl-1,2-propanediol. On the other hand, when exposed under the same conditions, methylamphetamine sulphate (MAS) remained virtually unchanged. Implications relating to evidence gathering for forensic purposes and to environmental assessment of clandestine drug laboratories are discussed.

Changes in microbial properties associated with long-term arsenic and DDT contaminated soils at disused cattle dip sites.

This work examined the effect of long-term arsenic (As) and DDT contamination on soil microbial properties at 11 cattle dip sites in northern New South Wales, Australia. Total As in the surface (0-10 cm) soils from these sites ranged from 34 to 2941 mg As kg(-1) soil and hexane-extractable DDT concentrations ranged between 2.9 and 7673 mg DDT kg(-1) soil. The concentrations of water and oxalate-extractable As were positively correlated with total As. Oxalate-extractable As was more strongly correlated (r(2)=0.87) with total As than water-extractable As (r(2)=0.34). A weak positive relationship was observed between the level of nutrient (organic carbon and nitrogen) and microbial biomass C (r(2)=0.61 and 0.45, respectively). There was a highly significant difference between the microbial properties of polluted and unpolluted sites (P<0.001). In comparison to unpolluted soils, fungal counts, microbial biomass C, and respiration were dramatically reduced (P<0.05) in polluted soils. However, the bacterial population between polluted and unpolluted soils were not different (P<0.05). The results of this study suggested that (a) long-term contamination of soils adjacent to former cattle dipping sites by As and DDT adversely affected soil microbial properties with the fungal populations being the most sensitive and (b) there was little regeneration of microbiota despite 25 years of field ageing of the soils.

Toxicity of hexavalent chromium and its reduction by bacteria isolated from soil contaminated with tannery waste.

An Arthrobacter sp. and a Bacillus sp., isolated from a long-term tannery waste contaminated soil, were examined for their tolerance to hexavalent chromium [Cr(VI)] and their ability to reduce Cr(VI) to Cr(III), a detoxification process in cell suspensions and cell extracts. Both bacteria tolerated Cr(VI) at 100 mg/ml on a minimal salts agar medium supplemented with 0.5% glucose, but only Arthrobacter could grow in liquid medium at this concentration. Arthrobacter sp. could reduce Cr(VI) up to 50 microg/ml, while Bacillus sp. was not able to reduce Cr(VI) beyond 20 microg/ml. Arthrobacter sp. was distinctly superior to the Bacillus sp. in terms of their Cr(VI)-reducing ability and resistance to Cr(VI). Assays with permeabilized (treated with toluene or Triton X 100) cells and crude extracts demonstrated that the Cr(VI) reduction was mainly associated with the soluble protein fraction of the cell. Arthrobacter sp. has a great potential for bioremediation of Cr(VI)-containing waste.

Bioavailability of an organophosphorus pesticide, fenamiphos, sorbed on an organo clay.

Hydrolysis of an insecticide/nematicide, fenamiphos [ethyl-3-methyl-4-(methylthio)phenyl-(1-methylethyl)phosphoramidate], immobilized through sorption by cetyltrimethylammonium-exchanged montmorillonite (CTMA-clay) by a soil bacterium, Brevibacterium sp., was examined. X-ray diffraction analysis, infrared spectra, and a negative electrophoretic mobility strongly indicated that fenamiphos was intercalated within the bacterially inaccessible interlayer spaces of CTMA-clay. The bacterium hydrolyzed, within 24 h, 82% of the fenamiphos sorbed by the CTMA-clay complex. There was a concomitant accumulation of hydrolysis product, fenamiphos phenol, in nearly stoichiometric amounts. During the same period, in abiotic (uninoculated) controls, 4.6% of the sorbed insecticide was released into the aqueous phase as compared to 6.0% of the sorbed fenamiphos in another abiotic control where activated carbon, a sink for desorbed fenamiphos, was present. Thus, within 24 h, the bacterium hydrolyzed 77% more fenamiphos sorbed by organo clay than the amounts desorbed in abiotic controls. Such rapid degradation of an intercalated pesticide by a bacterium has not been reported before. Evidence indicated that extracellular enzymes produced by the bacterium rapidly hydrolyzed the nondesorbable fenamiphos, even when the enzyme itself was sorbed. Fenamiphos strongly sorbed to an organo clay appears to be readily available for exceptionally rapid degradation by the bacterium.

Hydrolysis of fenamiphos and its oxidation products by a soil bacterium in pure culture, soil and water.

A bacterium, identified as Brevibacterium sp. MM1, readily hydrolysed fenamiphos, a widely used organophosphorus insecticide and its toxic oxides (fenamiphos sulfoxide, fenamiphos sulfone), which all contain a common P-O-C bond, in a mineral salts medium. The bacterium also hydrolysed fenamiphos and its oxides in soil and groundwater. Interestingly, fenamiphos phenol, fenamiphos sulfoxide phenol and fenamiphos sulfone phenol, formed during bacterial hydrolysis of fenamiphos and its oxides, persisted in the mineral salts medium, but were transitory in soil and groundwater due to their further metabolism by indigenous micro-organisms. The cell-free preparation (crude enzyme) of this bacterium was very effective in hydrolysing fenamiphos. This is the first report on exceptionally rapid hydrolysis of fenamiphos by a bacterium in pure cultures, soil and groundwater.

Influence of petroleum hydrocarbon contamination on microalgae and microbial activities in a long-term contaminated soil.

Petroleum hydrocarbons are widespread environmental pollutants. Although biodegradation of petroleum hydrocarbons has been the subject of numerous investigations, information on their toxicity to microorganisms in soil is limited, with virtually no work conducted on soil algae. We carried out a screening experiment for total petroleum hydrocarbons (TPH) and their toxicity to soil algal populations, microbial biomass, and soil enzymes (dehydrogenase and urease) in a long-term TPH-polluted site with reference to an adjacent unpolluted site. Microbial biomass, soil enzyme activity, and microalgae declined in medium to high-level (5,200-21,430 mg kg(-1) soil) TPH-polluted soils, whereas low-level (<2,120 mg kg(-1) soil) pollution stimulated the algal populations and showed no effect on microbial biomass and enzymes. However, inhibition of all the tested parameters was more severe in soil considered to have medium-level pollution than in soils that were highly polluted. This result could not be explained by chemical analysis alone. Of particular interest was an observed shift in the species composition of algae in polluted soils with elimination of sensitive species in the medium to high polluted soils. Also, an algal growth inhibition test carried out using aqueous eluates prepared from polluted soils supported these results. Given the sensitivity of algae to synthetic pollutants, alteration in the algal species composition can serve as a useful bioindicator of pollution. The results of this experiment suggest that chemical analysis alone is not adequate for toxicological estimations and should be used in conjunction with bioassays. Furthermore, changes in species composition of algae proved to be more sensitive than microbial biomass and soil enzyme activity measurements.

Effects of long-term contamination of DDT on soil microflora with special reference to soil algae and algal transformation of DDT.

DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and its principle metabolites, DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) and DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) are widespread environmental contaminants but little information is available concerning their effects on non-target microflora (especially microalgae and cyanobacteria) and their activities in long-term contaminated soils. For this reason a long-term DDT-contaminated soil was screened for DDT residues and toxicity to microorganisms (bacteria, fungi, algae), microbial biomass and dehydrogenase activity. Also, five pure cultures isolated from various sites (two unicellular green algae and three dinitrogen-fixing cyanobacteria) were tested for their ability to metabolise DDT. Viable counts of bacteria and algae declined with increasing DDT contamination while fungal counts, microbial biomass and dehydrogenase activity increased in medium-level contaminated soil (27 mg DDT residues kg(-1) soil). All the tested parameters were greatly inhibited in high-level contaminated soil (34 mg DDT residues kg(-1) soil). Species composition of algae and cyanobacteria was altered in contaminated soils and sensitive species were eliminated in the medium and high contaminated soils suggesting that these organisms could be useful as bioindicators of pollution. Microbial biomass and dehydrogenase activity may not serve as good bioindicators of pollution since these parameters were potentially influenced by the increase in fungal (probably DDT resistant) counts. All the tested algal species metabolised DDT to DDE and DDD; however, transformation to DDD was more significant in the case of dinitrogen-fixing cyanobacteria.

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to degradation by pure cultures of 1,1-diphenylethylene-degrading aerobic bacteria.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE). Biodegradation of DDE and DPE by bacteria has so far not been shown. Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE. Styrene-metabolizing bacteria (Rho-dococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy. Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl. This is the first report of the utilization of DPE as sole carbon and energy source by bacteria. All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates. DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation. The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria.

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) to cometabolic degradation by pure cultures of aerobic and anaerobic bacteria.

Pure cultures of aerobic and anaerobic bacteria capable of oxidation and reductive dehalogenation of chloroethylenes, and aerobic bacteria involved in biodegradation of polychlorinated biphenyls (PCBs) were screened for their ability to cometabolize the persistent pollutant 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE). Bacterial cultures expressing methane monooxygenase (Methylosinus trichosporium), propane monooxygenase (Mycobacterium vaccae) and biphenyl 2,3-dioxygenase enzymes (Pseudomonas fluorescens and Rhodococcus globerulus), as well as bacteria reductively dechlorinating chloroethylenes (Acetobacterium woodii and Clostridium butyricum) could not degrade DDE. Cell-free extracts of M. trichosporium, M. vaccae, P. fluorescens and R. globerulus were also unable to transform DDE, indicating that cell wall and membrane diffusion barriers were not biodegradation limiting. These studies suggest that these bacteria can not degrade DDE, even when provided with cosubstrates that induce chlorophenyl- and dichloroethylene-group transforming enzymes.