Remediation of copper-contaminated soil by Kocuria flava CR1, based on microbially induced calcite precipitation

An indigenous calcifying bacterial strain CR1, identified as Kocuria flava, was isolated from soil of a mining area, Urumqi, China. An extensive copper bioremediation capacity of this isolate was studied based on microbially induced calcite precipitation (MICP). K. flava CR1 removed 97% of copper when initial Cu concentration was 1000 mg L⁻¹. The isolate produced significant amount of urease (472 U mL⁻¹), an enzyme that leads to calcite precipitation. The isolate removed 95% of copper from contaminated soil. The MICP process in bioremediation was further confirmed by FTIR and XRD analyses. FTIR analysis showed two different forms of calcium carbonate, i.e., calcite and aragonite, and the results were well supported by XRD. For the first time, the ability of K. flava has been documented in the bioremediation of polluted soil. This study showed that MICP-based bioremediation by K. flava is a viable, environmental friendly technology for cleaning-up the copper-contaminated site.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Growth and physiological response of lemongrass (Cymbopogon citratus (D.C.) Stapf.) under different levels of fly ash-amended soil

Revegetation with metal tolerant plants for management of fly ash deposits is an important environmental perspective nowadays. Growth performance, photosynthesis, and antioxidant defense of lemongrass (Cymbopogon citratus (D.C.) Stapf.) were evaluated under various combination of fly ash amended with garden soil in order to assess its fly ash tolerance potential. Under low level of fly ash (25%) amended soil, the plant growth parameters such as shoot, root, and total plant biomass as well as metal tolerance index were increased compared to the control plants grown on garden soil, followed by decline under higher concentration of fly ash (50%, 75% and 100%). In addition, leaf photosynthetic rate, stomatal conductance, and photosystem (PS) II activity were not significantly changed under low level of fly ash (25%) amended soil compared to the garden soil but these parameters were significantly decreased further with increase of fly ash concentrations. Furthermore, increase of activities of some antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and guaiacol peroxidase over control were noticed in lemongrass under all fly ash treatments. Taken together, the study suggests that lemongrass can be used for phytoremediation of fly ash at 25% amended soil.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Differential expression of CYP102 in Bacillus megaterium by 17-beta-estradiol and 4-sec-butylphenol

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.     

Cyclodextrin glucosyltransferase production by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> NCR: evaluation and optimization of culture conditions using factorial design

Statistically-based experimental designs were used to optimize the production of cyclodextrin glucosyltransferase (CGTase) from a local isolate of Bacillus megaterium using shack culture fermentation. Seven cultural conditions were examined for enzyme production and specific activity using Plackett-Burman factorial design. Fermentation time and K₂HPO₄ level were the crucial for factors improving enzyme production process. The steepest ascent design was adopted-based on the results recorded with Plackett-Burman design. Maximal enzyme estimates (activity 56.1 U/ml, and specific activity 62.7 U/mg protein) were achieved. A verification experiment was carried out to examine model validation of this optimization.     

Is Vigna radiata suitable for the revegetation of fly ash landfills?

The present article briefly describes the concerns regarding the suitability of Vigna radiata L. for the revegetation of fly ash landfills.     

From Metchnikoff to Monsanto and beyond: the path of microbial control

In 125 years since Metchnikoff proposed the use of Metarhizium anisopliae to control the wheat cockchafer and brought about the first field trials, microbial control has progressed from the application of naturalists' observations to biotechnology and precision delivery. This review highlights major milestones in its evolution and presents a perspective on its current direction. Fungal pathogens, the most eye-catching agents, dominated the early period, but major mycological control efforts for chinch bugs and citrus pests in the US had questionable success, and interest waned. The discoveries of Bacillus popilliae and Bacillus thuringiensis began the era of practical and commercially viable microbial control. A program to control the Japanese beetle in the US led to the discovery of both B. popilliae and Steinernema glaseri, the first nematode used as a microbial control agent. Viral insect control became practical in the latter half of the 20th century, and the first registration was obtained with the Heliothis nuclear polyhedrosis virus in 1975. Now strategies are shifting for microbial control. While Bt transgenic crops are now planted on millions of hectares, the successes of more narrowly defined microbial control are mainly in small niches. Commercial enthusiasm for traditional microbial control agents has been unsteady in recent years. The prospects of microbial insecticide use on vast areas of major crops are now viewed more realistically. Regulatory constraints, activist resistance, benign and efficacious chemicals, and limited research funding all drive changes in focus. Emphasis is shifting to monitoring, conservation, integration with chemical pesticides, and selection of favorable venues such as organic agriculture and countries that have low costs, mild regulatory climates, modest chemical inputs, and small scale farming.     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Production of extracellular polysaccharide by Bacillus megaterium RB-05 using jute as substrate

Bacillus megaterium RB-05 was grown on glucose and on “tossa-daisee” (Corchorus olitorius)-derived jute, and production and composition of extracellular polysaccharide (EPS) were monitored. An EPS yield of 0.065 ± 0.013 and of 0.297 g ± 0.054 g⁻¹ substrate after 72 h was obtained for glucose and jute, respectively. EPS production in the presence of jute paralleled bacterial cellulase activity. High performance liquid chromatography (HPLC), matrix assisted LASER desorption/ionization-time of flight (MALDI-ToF) mass spectroscopy, and fourier transform infrared (FT-IR) spectroscopy demonstrated that the EPS synthesized in jute culture (JC) differed from that synthesized in glucose mineral salts medium (GMSM). While fucose was only a minor constituent (4.9 wt.%) of EPS from GMSM, it a major component (41.9 wt.%) of EPS synthesized in JC. This study establishes jute as an effective fermentation substrate for EPS production by a cellulase-producing bacterium.     

Chemical,microbial and physical properties of manufactured soils produced by co-composting municipal green waste with coal fly ash

Increasing proportions of coal fly ash were co-composted with municipal green waste to produce manufactured soil for landscaping use. Only the 100% green waste treatment reached a thermophilic composting phase (?50 °C) which lasted for 6 days. The 25% and 50% ash treatments reached 36–38 °C over the same period while little or no self-heating occurred in the 75% and 100% ash treatments. Composted green waste had a low bulk density and high total and macro-porosity. Addition of 25% ash to green waste resulted in a 75% increase in available water holding capacity. As the proportions of added ash in the composts increased, the organic C, soluble C, microbial biomass C, basal respiration and activities of β-glucosidase, L-asparaginase, alkali phosphatase and arylsulphatase enzymes in the composted products all decreased. It could be concluded that addition of fly ash to green waste at a proportion higher than 25% did not improve the quality parameters of manufactured soil.     

Phosphate Solubilizing Bacillus megaterium mj1212 Regulates Endogenous Plant Carbohydrates and Amino Acids Contents to Promote Mustard Plant Growth

The current study was conducted to explore the potential of a phosphate solubilizing soil bacterium, Bacillus megaterium mj1212 for enhancing the growth of mustard plants. The newly isolated bacterial strain mj1212 was identified as B. megaterium using phylogenetic analysis and, its phosphate solubilization ability was shown by the clear zone formation on National Botanical Research Institute’s Phosphate medium. Moreover, the phosphate solubilization ability of B. megaterium mj1212 was enhanced by optimal culture conditions at pH 7.0 and 35 °C which might be due to the presence of malic and quinic acid in the culture medium. The beneficial effect of B. megaterium mj1212 in mustard plants was determined by an increasing shoot length, root length and fresh weight of plants. In the biochemical analysis revealed that chlorophyll, sucrose, glucose, fructose and amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ilu, Leu, Tyr, Phe, Lys, His, Arg and Pro) were higher in B. megaterium mj1212 treated plants, when compared to their control. The result of present study suggests that B. megaterium mj1212 treatment could be act as phosphate biofertilizer to improve the plant growth.     

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations

Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.     

The host range of the male-killing symbiont Arsenophonus nasoniae in filth fly parasitioids

The Son-killer bacterium, Arsenophonus nasoniae, infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This gammaproteobacterium kills a substantial amount of male embryos produced by an infected female. Aside from male death, the bacterium does not measurably affect the host, and how it is maintained in the host population is unknown. Interestingly, this bacterial symbiont can be transmitted both vertically (from mother to offspring) and horizontally (to unrelated Nasonia wasps developing in the same fly host). This latter mode may allow the bacterium to spread throughout the ecological community of filth flies and their parasitoids, and to colonize novel species, as well as permit its long-term persistence.We tested 11 species of filth flies and 25 species of their associated parasitoids (representing 28 populations from 16 countries) using diagnostic PCR to assess the bacterium’s actual host range. In addition to 16S rRNA, two loci were targeted: the housekeeping gene infB, and a sequence with high homology to a DNA polymerase gene from a lysogenic phage previously identified from other insect symbionts. We identified infections of A. nasoniae in four species of parasitoids, representing three taxonomic families. Highly similar phage sequences were also identified in three of the four species. These results identify the symbiont as a generalist, rather than a specialist restricted solely to species of Nasonia, and also that horizontal transmission may play an important role in its maintenance.     

Trichomycete symbiotes of Crozetia seguyi, a primitive black fly

Crozetia is a genus of black flies endemic to the Crozet Islands in the Indian Ocean. No internal symbiotes were previously known from Crozetia species. We report two species of trichomycete symbiotes Stachylina litoralis and Smittium culicisoides from Crozetia seguyi. Larvae of C. seguyi were examined from three sites. The infection rates for St. litoralis was 10.0-33.3% (n=47) of the larvae and Sm. culicisoides was 46.1-85.7% (n=47). No other symbiotes were discovered.