Degradation of chlorpyriphos and polyethylene by endosymbiotic bacteria from citrus mealybug

Chlorpyriphos is one of the major organophosphorus pesticides used widely to control a range of insect pests across several crops. This insecticide is hazardous to the environment and toxic to mammals, thus, it is essential to remove the same from the environment. Similarly, use of polythene is also increasing day by day. Therefore, it is highly important to identify ways to degrade chlorpyriphos and other pesticides from the environment. We studied the degradation of chlorpyriphos and polyethylene by Citrus mealybug (Planococcus citri) bacterial endosymbionts such as Bacillus licheniformis, Pseudomonas cereus, Pseudomonas putida and Bacillus subtilis. This investigation revealed that bacterial endosymbionts use the polythene as a source of carbon and solubilize them by their enzymatic machinery. The degradation of polyethylene by endosymbionts showed a significant reduction in weight of polyethylene sheet after 15, 30 and 45 days of treatment. The SEM images showed localized degradation of the polyethylene around the bacterial cells in the biofilm. Further, the tensile strength (percentage elongation) was significantly reduced after 45 days of incubation. The weight of paraffin wax showed significant reduction in B. cereus. A significant reduction in total amount of chlorpyriphos in soil was observed at an interval of 7, 14 and 21 days after treatment by the bacterial isolates. Among the bacteria, B. cereus and P. putida were found to be most effective. The results from this study show that endosymbionts can be significantly implicated in degrading chlorpyriphos and polyethylene from the environment.     

Antagonist effects of strains of Bacillus spp. against Rhizoctonia solani for their protection against several plant diseases: Alternatives to chemical pesticides

Rhizoctonia solan Kühn (teleomorph: Thanatephorus cucumeris (Frank) Donk (R. solani) is a soil-borne phytopathogenic species complex as well as a necrotic fungus that causes significant crop yield losses worldwide. Agronomic practices (crop rotation), resistant cultivars, and chemical pesticides are widely used to control R. solani. However, these practices are insufficient to control the pest. Moreover, the application of chemical pesticides is harmful to both the environment and human health. Therefore, the use of biocontrol agents (BCAs) and that of plant-growth promoting bacteria (PGPB) are considered to be potentially sustainable, cost-effective, efficient, and eco-friendly ways to control R. solani. Several microorganisms have been used as biocontrol agents (BCAs) to manage R. solani. Among these, biocontrol agents (BCAs) Bacillus spp. are used to promote plant growth. Furthermore, due to its broad range of antibiotic-producing abilities, Bacillus spp. is widely used against R. solani. In this review, current and previous studies about the ability of Bacillus spp. to control diseases caused by R. solani are reported. It also focuses on the plant-growth promotion attributes of Bacillus spp. in response to the deleterious effects of R. solani.     

Carbamate and Organophosphorus Nematicides: Acetylcholinesterase inhibition and Effects on Dispersal

The sensitivities of acetylcholinesterases (ACHE) from the fungus-feeder Aphelenchus avenae and the plant-parasitic species Helicotylenchus dihystera and Pratylenchus penetrans and the housefly, Musca domestica, were compared using a radiometric assay which utilized H³ acetylcholine as a substrate. Nematode ACHE were generally less sensitive to inhibition by organophosphorns and carbamate pesticides than were ACHE from the housefly. ACHE from the plant-parasitic species and A. avenae were generally similar in sensitivity. In soil, carbamates were more toxic than the organophosphorus pesticides to A. avenae. All pesticides tested affected nematode movement, but fenamiphos was more inhibitory than others. The effects on dispersal of nematodes may be an important mechanism in control by some nematicides.     

Design,synthesis, in silico studies and in vitro evaluation of isatin-pyridine oximes hybrids as novel acetylcholinesterase reactivators

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.     

Organophosphorus and Organochlorine Pesticides Bioaccumulation by Eichhornia crassipes in Irrigation Canals in an Urban Agricultural System

A natural wetland in Mexico City Metropolitan Area is one of the main suppliers of crops and flowers, and in consequence its canals hold a high concentration of organochlorine (OC) and organophosphorus (OP) pesticides. There is also an extensive population of water hyacinth (Eichhornia crassipes), which is considered a plague; but literature suggests water hyacinth may be used as a phytoremediator. This study demonstrates bioaccumulation difference for the OC in vivo suggesting their bioaccumulation is ruled by their log K_ow, while all the OP showed bioaccumulation regardless of their log K_ow. The higher bioaccumulation factors (BAF) of the accumulated OC pesticides cannot be explained by their log K_ow, suggesting that the OC pesticides may also be transported passively into the plant. Translocation ratios showed that water hyacinth is an accumulating plant with phytoremediation potential for all organophosphorus pesticides studied and some organochlorine pesticides. An equation for free water surface wetlands with floating macrophytes, commonly used for the construction of water-cleaning wetlands, showed removal of the pesticides by the wetland with room for improvement with appropriate management.     

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC₅₀ of 4.17×10⁻⁸ M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.     

Comparison of in-situ biodegrading abilities of Pseudomonas putida mutants: leuB− auxotroph,fliC− non-motility,and cheA− non-chemotaxis

Bioremediation of pollutants in natural environments is affected by many factors, such as bacterial survival, motility, and chemotaxis. However, these roles in in-situ biodegradation of organophosphorus pesticides have not been examined extensively. In this paper, a highly effective methyl-parathion (MP) degrading strain, Pseudomonas putida DLL-1, which also demonstrates motile ability and chemotactic response toward MP, was selected as the research material. A leuB^? auxotroph mutant A3-27 and fliC^? non-motility mutant a4-8 were first constructed by random insertion of the kanamycin gene into the chromosome of P. putida DLL-1 with the mini-transposon system. Biodegradation of MP in liquid medium and soil microcosms by A3-27, a4-8 and a previously constructed cheA^? non-chemotaxis mutant P. putida DAK were compared. The kinetic parameters for MP degradation were all similar in the well-mixed liquid systems. However, in soil microcosms, all the three mutants had lower degrading rates compared with wild-type P. putida DLL-1. The auxotroph mutant A3-27 had the lowest degrading rate and could only degrade 25.7–34.2% MP in 5 days, and the non-motility mutant a4-8 and non-chemotaxis mutant DAK could only degrade 53.5–68.1% and 64.3–85.7% MP, respectively. This paper emphasizes, for the first time, the use of non-auxotroph bacteria for efficient removal of organophosphorus pesticides in contaminated sites, and also points out the importance of select microorganisms with specific motile or chemotactic affinities in optimizing pesticide bioremediation.     

Surface-Active Agents from Two Bacillus Species

Two Bacillus species were studied which produced bioemulsifiers; however, they were distinctly different compounds. Bacillus sp. strain IAF 343 produced unusually high yields of extracellular biosurfactant when grown on a medium containing only water-soluble substrates. The yield of 1 g/liter was appreciably better than those of most of the biosurfactants reported previously. This neutral lipid product, unlike most lipid biosurfactants, had significant emulsifying properties. It did not appreciably lower the surface tension of water. On the same medium, Bacillus cereus IAF 346 produced a more conventional polysaccharide bioemulsifier, but it also produced a monoglyceride biosurfactant. The bioemulsifier contained substantial amounts of glucosamine and originated as part of the capsule layer. The monoglyceride lowered the surface tension of water to 28 mN/m. It formed a strong association with the polysaccharide, and it was necessary to use ultrafiltration to effect complete separation. The removal of the monoglyceride caused the polysaccharide to precipitate. It is suggested that earlier reports of biopolymers which both stabilized emulsions and lowered surface tension were actually similar aggregates of lipid and bioemulsifier.     

Production of Diarrheal Enterotoxins and Other Potential Virulence Factors by Veterinary Isolates of Bacillus Species Associated with Nongastrointestinal Infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Investigation of antibacterial activity of Bacillus spp. isolated from the feces of Giant Panda and characterization of their antimicrobial gene distributions

Bacillus group is a prevalent community of Giant Panda’s intestinal flora, and plays a significant role in the field of biological control of pathogens. To understand the diversity of Bacillus group from the Giant Panda intestine and their functions in maintaining the balance of the intestinal microflora of Giant Panda, this study isolated a significant number of strains of Bacillus spp. from the feces of Giant Panda, compared the inhibitory effects of these strains on three common enteric pathogens, investigated the distributions of six universal antimicrobial genes (ituA, hag, tasA, sfp, spaS and mrsA) found within the Bacillus group by PCR, and analyzed the characterization of antimicrobial gene distributions in these strains using statistical methods. The results suggest that 34 strains of Bacillus spp. were isolated which has not previously been detected at such a scale, these Bacillus strains could be classified into five categories as well as an external strain by 16S rRNA; Most of Bacillus strains are able to inhibit enteric pathogens, and the antimicrobial abilities may be correlated to their categories of 16S rRNA; The detection rates of six common antimicrobial genes are between 20.58 %(7/34) and 79.41 %(27/34), and genes distribute in three clusters in these strains. We found that the antimicrobial abilities of Bacillus strains can be one of the mechanisms by which Giant Panda maintains its intestinal microflora balance, and may be correlated to their phylogeny.     

Phenotyping using semi-automated BIOLOG and conventional PCR for identification of Bacillus isolated from biofilm of sink drainage pipes

The presence of Bacillus in natural biofilms which develop in sink drainage pipes is not widely studied. Therefore, the main aim of this study was to isolate and identify Bacillus spp. using the BIOLOG GEN III system as a phenotypic fingerprint and polymerase chain reaction (PCR). A total of 61 biofilms samples were collected from sink drainage pipes in a kitchen and bathroom of different households in Helwan area and both laboratory and hospital collected from National Research Centre (NRC). Bacillus was isolated from the biofilms using HiCrome Bacillus Agar followed by isolates identification by both BIOLOG to the species level and PCR using genus specific primers to the genera level. Bacillus was detected in all tested biofilm samples (61 samples). The highest counts were observed in hospital sink drainage pipes (10⁵?CFU/10?cm²) while; the lowest counts were observed in both bathroom and laboratory sink drainage pipes (10²?CFU/10 cm^?2). In total, 61% Bacillus isolates were identified by BIOLOG while, 67% isolates were confirmed by PCR. The diversity of Bacillus among species level using BIOLOG were 34% B. cereus, 23% B. subtilis ss subtilis, 17% B. thuringiensis, 16% B. licheniformis and 13% B. amyloliquefaciens. It can be concluded that; PCR is more sensitive than BIOLOG for identification of Bacillus. However, BIOLOG can identify Bacillus at species level and test 94 carbon and chemical sources on a microplate in one shot. Thus, the combination between phenotyping by BIOLOG and molecular approaches such as PCR for identification of bacterial isolates is recommended.     

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments – atropine, oximes and diazepam – are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested.     

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.     

乙氧基磷酸酯类有机磷农药单克隆抗体的制备与鉴定

制备针对乙氧基磷酸酯类有机磷农药的单克隆抗体,以此为基础建立该类农药的快速免疫筛选检测方法．以二乙基磷酸乙酸为通用结构半抗原,分别使之与牛血清白蛋白和鸡卵清蛋白共价偶联,合成免疫原和包被原并对其进行结构鉴定．偶联成功后的免疫原用于免疫Balb/c小鼠．将免疫成功小鼠的脾细胞与小鼠SP2/0骨髓瘤细胞融合,筛选能稳定分泌抗乙氧基磷酸酯类有机磷农药单克隆抗体的杂交瘤细胞株．获得的小鼠腹水用辛酸-硫酸铵法纯化,所得纯化抗体以琼脂双扩散法鉴定其免疫球蛋白类型,间接竞争ELISA方法测定其对半抗原的灵敏度、特异性和亲和性．结果表明,该抗体分泌IgG1亚类的单克隆抗体,且与二乙基磷酸乙酸的亲和性较高(1.4×10⁷ L/mol),所得抗体对毒死蜱、对硫磷、丙溴磷、氧化乐果、除线磷、二嗪农、溴硫磷、辛硫磷、喹硫磷、三唑磷等农药有特异性反应．该检测技术可用于上述农药的快速定性或定量检测．     

Production of lipopeptides among <Emphasis Type="Italic">Bacillus</Emphasis> strains showing growth inhibition of phytopathogenic fungi

The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance to eliminate chemical pollutants.     

ε-PolyLysine production from sugar cane molasses by a new isolates of Bacillus sp. and optimization of the fermentation condition

An attempt was made to use cane molasses as a culture medium for ε-PolyLysine (ε-PL) production by a natural bacterial isolate. The bacterium was identified as Bacillus sp., as confirmed by 16S rDNA sequence analysis. A BLAST result of the sequence indicated that the closest relative of this Bacillus BHU strain was B. thuringiensis, with 97 % homology. The molasses was found to be a better culture medium compared to commonly used culture media comprised of either glucose or glycerol as a carbon source. The various physicochemical parameters were studied for culture growth and polymer production, and were further optimized using response surface methodology (RSM). The correlation coefficient of the resulting model was found to be R ²?=?0.9828. The RSM predicted optimum conditions for ε-PL production (2.46 g/l) by the Bacillus strain was achieved by using molasses, 59.7 g/l; yeast extract, 15.2 mg/l; pH, 6.8 and fermentation time, 42 h at 30 °C. This study represents the first report on the potential application of cane molasses (a byproduct of sugarcane industries) as a culture medium for ε-PL production by Bacillus species. The specific Bacillus strain used in the present study can be exploited for developing a novel technology using inexpensive renewable resources for ε-PL production, a polymer of commercial interest.     

鼠李糖乳杆菌碱性磷酸酶的提取条件优化及其降解有机磷农药作用

【背景】我国作为农业大国,对农药的大量使用是不可避免的,但是农药的超范围使用、超标及高检出率对于环境的污染与人体健康的威胁日趋严重。【目的】碱性磷酸酶(alkaline phosphatase,ALP)对有机磷农药具有积极的降解作用,因此,本文对鼠李糖乳杆菌(Lactobacillus rhamnosus) Z23(LGG Z23)所产碱性磷酸酶的提取条件进行优化,并研究其对有机磷农药的降解作用。【方法】使用单因素试验和正交试验优化ALP的提取条件;使用对硝基苯酚法测定酶活力;使用分级沉淀和层析法提纯ALP;使用乙酰胆碱酯酶抑制法测定ALP对有机磷农药的降解率。【结果】LGG Z23所产ALP的最优提取条件为：细胞破碎时间15 min,破碎功率450 W,料液比(质量体积比)1:6,提取液pH 10.0,此条件下酶活力为(4.95±0.26) U/mL,比优化前提高2.11倍;对6种有机磷农药的降解率效果为敌敌畏(95.79%±0.01%)>甲基对硫磷(90.69%±0.03%)>毒死蜱(88.90%±0.02%)>敌百虫(86.07%±0.03%)>马拉硫磷(85.31%±0.02%)>乐果(83.18%±0.03%),其中对敌敌畏和甲基对硫磷的降解效果最好,可达90%以上,并且降解作用差异显著(P<0.05)。【结论】本研究为LGG Z23所产ALP的应用研究提供了理论依据和实验数据。     

Endospore production of Bacillus spp. for industrial use

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.