PCR-RAPD profiling of Fusarium spp. causing guava wilt disease in India

Wilt is a serious disease of the guava crop in India. Fusarium oxysporum f. sp. psidii and F. solani have been reported as causative agents of this disease. In this study, 42 isolates each of F. oxysporum f. sp. psidii and F. solani, were isolated from guava cultivars and characterized by randomly amplified polymorphic DNA (RAPD) method. Thirty RAPD primers were tested in the genome of Fusarium spp. and the number of scorable bands for corresponding primer ranged from 1-8 with an average of 5 bands per individual. DNA band size ranged from 200 bp to 5090 bp. A 0.21 per cent polymorphism was found in individual isolates of F. solani indicating that the 42 isolates were similar. However, a 2.58 percent polymorphism among individual isolates of F. oxysporum f.sp. psidii showed a higher level of genetic diversity. Cluster analysis of the RAPD band patterns clearly separated the isolates of F. oxysporum f.sp. psidii into three clusters. Two clusters were formed with F. solani isolates, showing a higher degree of similarity. Unique fingerprint profiles generated by the PCR-RAPD can be exploited for genetic characterization purposes.     

Physical-chemical and ecotoxicological evaluation of water based drilling fluids used in Italian off-shore

In order to evaluate the effects on the marine ecosystem caused by an eventual discharge into sea of water based drilling fluids, as current legislation allows, chemical and ecotoxicological analyses were performed on the most common drilling muds and products used in Italian off-shore activities. The chemical analysis on drilling fluids involved the leaching test and the measurement of total content of heavy metals, whereas biodegradation tests were performed on the products used in mud's formulations. As for ecotoxicological evaluation, two marine organisms, the crustacean Artemia salina and the diatom Phaeodactylum tricornutum, were selected to determine the LC₅₀ and the EC₅₀ respectively.     

Relationship between chromium(VI) resistance and extracellular polymeric substances (EPS) concentration by some cyanobacterial isolates

BACKGROUND, AIM, AND SCOPE: Chromium(VI) resistance and its association with extracellular polymeric substance (EPS) concentration in cyanobacteria was investigated. Increased EPS concentration was associated with Cr(VI) resistance. The most resistant isolate, Chroococcus sp. H(4), secreted the most EPS (427 mg/L). MATERIALS AND METHODS: EPS concentration of the two most resistant isolates (Chroococcus sp. H(4) and Synechocystis sp. S(63)) was investigated following exposure to 15 and 35 ppm Cr(VI). The composition of EPS produced by Chroococcus sp. H(4) following exposure to 10 ppm Cr(VI) was analyzed using high-performance liquid chromatography. Control EPS was composed of glucose (99%) and galactronic acid (1%); in the presence of 10 ppm Cr(VI), EPS composition changed to glucose (9%), xylose (75%), rhamnose (14%), and galacturonic acid (2%). RESULTS AND DISCUSSION: Results indicated that (1) exposure to elevated concentrations of Cr(VI) affected the composition of EPS produced by Chroococcus sp. H(4), and (2) there was a correlation between Cr(VI) resistance and EPS concentration in some cyanobacteria.     

Degradation and metabolism of hexazinone by two isolated bacterial strains from soil

Two hexazinone-degrading bacterial strains were isolated from soil by enrichment culture technique, and identified as Pseudomonas sp. and Enterobacter cloacap. The two purified isolates, designated as WFX-1 and WFX-2, could rapidly degrade hexazinone with a half-life of 3.08 days and 2.95 days in mineral salt medium (MSM), while their mixed bacterial culture was found to degrade hexazinone, at an initial concentration of 50 microg/ml, by enhancing 2.3-fold over that when the isolates were used alone. Two microbial metabolites (A and D) were obtained by preparative TLC and identified on the basis of the spectral data of IR, 1H NMR and HPLC-ESI-MS, but both of them were known products as they had been reported in soil and vegetation metabolites of hexazinone. However, metabolites B and C were new degradates, whose molecular weights (MW) were 157 and 156, respectively, being reported from microbial metabolism for the first time.     

Complete degradation of butyl benzyl phthalate by a defined bacterial consortium: role of individual isolates in the assimilation pathway

Two bacterial strains, in consortium, were isolated by enrichment techniques from municipal waste-contaminated soil, which utilized butyl benzyl phthalate (BBP) as the sole carbon source. One of the isolates was identified as Arthrobacter sp. strain WY and the other one as Acinetobacter sp. strain FW based on the morphological, nutritional and biochemical characteristics and 16S rRNA sequence analysis. Various metabolites of BBP engendered by Arthrobacter sp. strain WY were isolated and identified by a combination of chromatographic and spectrophotometric analyses, which revealed a pathway involving monobutylphthalate (MBuP), monobenzyl phthalate (MBzP), phthalic acid and protocatechuic acid. The protocatechuic acid in turn was processed by ortho-cleavage dioxygenase to form beta-carboxy-cis,cis-muconate, ultimately leading to the TCA cycle. The Arthrobacter sp. strain WY could not utilize the hydrolyzed alcohols of BBP. On the other hand, the Acinetobacter sp. strain FW, which by itself could not utilize BBP as the sole carbon source, is capable of utilizing the hydrolyzed alcohols of BBP. Benzyl alcohol was found to be metabolized by the Acinetobacter sp. strain FW via benzaldehyde, benzoic acid and catechol. Catechol was further degraded by ortho-cleavage dioxygenase to cis,cis-muconic acid and subsequently to muconolactone leading to beta-ketoadipate pathway. Moreover, the Acinetobacter sp. strain FW metabolized 1-butanol through butyraldehyde and butyric acid leading to the tricarboxylic acid cycle via beta-oxidation pathway. This is the first report on the complete degradation of BBP by a defined consortium describing the role of its individual constituents in the BBP assimilation pathway.     

A2/O工艺缺氧池中反硝化聚磷菌的比例、特性研究及菌株鉴定

用释磷/聚磷装置和微生物筛选、分离方法研究A2/O工艺缺氧池污泥,确定缺氧池中反硝化聚磷菌(DPB)的比例,筛选、分离得反硝化聚磷单菌株且对单菌株聚磷特性进行研究.结果表明,缺氧池中DPB占聚磷菌(PAO)的比例约为21.5%.从缺氧池分离得到的肠杆菌科、气单胞菌属和假单胞菌属都是DPB,而不动杆菌属仅是好氧PAO,葡萄球菌属和微球菌属仅是一种专职的反硝化菌.反应过程中同时存在O2和NO3时,肠杆菌科优先利用水中的O2进行聚磷;在缺氧环境中,肠杆菌科在COD为30mg/L时的聚磷效果优于COD为180 mg/L时的聚磷效果.可见DPB的反硝化和聚磷的特性与电子受体的存在形式和COD有密切关系.因此,改良传统A2/O工艺和研发同步反硝化聚磷装置时,必须控制缺氧反硝化聚磷单元中混合液的DO和COD.     

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

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

In vitro Cr(VI) reduction by cell-free extracts of chromate-reducing bacteria isolated from tannery effluent irrigated soil

Four efficient Cr(VI)-reducing bacterial strains were isolated from rhizospheric soil of plants irrigated with tannery effluent and investigated for in vitro Cr(VI) reduction. Based on 16S rRNA gene sequencing, the isolated strains SUCR44, SUCR140, SUCR186, and SUCR188 were identified as Bacillus sp. (JN674188), Microbacterium sp. (JN674183), Bacillus thuringiensis (JN674184), and Bacillus subtilis (JN674195), respectively. All four isolates could completely reduce Cr(VI) in culture media at 0.2 mM concentration within a period of 24–120 h; SUCR140 completely reduced Cr(VI) within 24 h. Assay with the permeabilized cells (treated with Triton X-100 and Tween 80) and cell-free assay demonstrated that the Cr(VI) reduction activity was mainly associated with the soluble fraction of cells. Considering the major amount of chromium being reduced within 24–48 h, these fractions could have been released extracellularly also during their growth. At the temperature optima of 28 °C and pH?7.0, the specific activity of Cr(VI) reduction was determined to be 0.32, 0.42, 0.34, and 0.28 μmol Cr(VI)?min^?1?mg^?1 protein for isolates SUCR44, SUCR140, SUCR186, and SUCR188, respectively. Addition of 0.1 mM NADH enhanced the Cr(VI) reduction in the cell-free extracts of all four strains. The Cr(VI) reduction activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg²⁺. Beside this, urea and thiourea also reduced the activity of chromate reduction to significant levels.     

含油钻屑高效除油剂及除油机理研究

油基钻井液在复杂井、深井的钻井作业中受到越来越多的使用和重视,但相应也产生了大量的废弃含油钻屑,带来了环境污染问题。针对含油钻屑的特点,研制了一种高效除油剂HBS-6,在阴离子表面活性剂、非离子表面活性剂、溶剂和软水剂等组分的协同作用下,利用强化分离设备,实现了钻屑含油的去除与无害化处置,含油钻屑除油率达90%以上。同时,还采用红外光谱、气相色谱和液相色谱等分析手段,研究了除油剂HBS-6的作用机理。HBS-6含有HLB值高且能生物降解的阴离子表面活性剂,以保证除油剂的清洗能力和环保性,同时还采用HLB值低的非离子表面活性剂,以提高对油的乳化和除油能力,为含油钻屑的无害化处理拓展了一条新的技术途径。     

Enrichment and isolation of a mixed bacterial culture for complete mineralization of endosulfan

In the present study, we isolated three novel bacterial species, namely, Staphylococcus sp., Bacillus circulans-I, and Bacillus circulans-II, from contaminated soil collected from the premises of a pesticide manufacturing industry. Batch experiments were conducted using both mixed and pure cultures to assess their potential for the degradation of aqueous endosulfan in aerobic and facultative anaerobic condition. The influence of supplementary carbon (dextrose) source on endosulfan degradation was also examined. After four weeks of incubation, mixed bacterial culture was able to degrade 71.82 +/- 0.2% and 76.04 +/- 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively, with an initial endosulfan concentration of 50 mg l(-1). Addition of dextrose to the system amplified the endosulfan degradation efficiency by 13.36 +/- 0.6% in aerobic system and 12.33 +/- 0.6% in facultative anaerobic system. Pure culture studies were carried out to quantify the degradation potential of these individual species. Among the three species, Staphylococcus sp. utilized more beta endosulfan compared to alpha endosulfan in facultative anaerobic system, whereas Bacillus circulans-I and Bacillus circulans-II utilized more alpha endosulfan compared to beta endosulfan in aerobic system. In any of these degradation studies no known intermediate metabolites of endosulfan were observed.     

Bioremediation of kerosene I: A case study in liquid media

The ability of different local isolates in addition to some isolates from Germany to degrade kerosene in liquid medium was studied. The results showed that the percent of kerosene degradation varied among the different organisms and that 59–94% of kerosene was degraded after 21 d. Two local isolates (Pseudomonas sp. AP and Pseudomonas sp. CK) and one German isolate (Gordonia sp. DM) were selected for this study. The addition of wheat bran, as co-substrate, stimulated the kerosene degradation by the two local strains, while glucose inhibited the degradation rate using the three organisms with different rates. Ammonium nitrate and urea was the best nitrogen sources. The use of superphosphate (as phosphorus source) in the presence of urea stimulates the degradation rate. It was also observed that the addition of 1% surfactants, like Triton X-100, Igepal, Tergitol, or Tween 20 and 80 enhanced the kerosene degradation. The degradation percent lied between 94% and 98%. The ability of the tested organisms to degrade kerosene concentration from 2% to 8% was evaluated. It was found that the three organisms degraded about 65–85% from 8% kerosene after 21 d. The use of rice straw-immobilized cells reduced the time of degradation and enhanced the degradation ability of the organisms. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the presence of a common protein band when the tested organisms were grown on kerosene.     

Reduction of perchlorate and nitrate by salt tolerant bacteria

Spent regenerant brine from ion-exchange technology for the removal of perchlorate and nitrate produces a high salt waste stream, which requires remediation before disposal. Bioremediation is an attractive treatment option. In this study, we enriched for salt tolerant bacteria from sediments from Cargill salt evaporation facility (California, USA), the Salton Sea (California, USA), and a high density hydrocarbon oxidizing bacterial cocktail. The bacterial cocktail enrichment culture reduced ClO4- from 500 to 260 mg 1 in 4 weeks. Salt tolerant bacterial isolates from the enrichment cultures and two denitrifying salt tolerant bacteria, Haloferax denitrificans and Parococcus halodenitricans, substantially reduced perchlorate. The highest rate of perchlorate removal was recorded with the isolate, Citrobacter sp.: 32% reduction in 1 week. This bacterium substantially reduced perchlorate in 0-5% NaCl solutions and maximally at 30 degrees C and at an initial pH 7.5. In simulated brines containing 7.5% total solids, the Citrobacter sp. significantly reduced both perchlorate and nitrate with 34.9 and 15.6% reduction, respectively, in 1 week. Coculture of a potent perchlorate reducing, non-salt tolerant (non-saline) bacterium, perclace and the Citrobacter sp. proved most effective for perchlorate removal in the brine (46.4% in 1 week). This study demonstrates that both anions can be reduced in treatment of brines from ion exchange systems.     

Potentiality of different isolates of wilt fungus Fusarium oxysporum collected from rhizosphere of tomato against root-knot nematode Meloidogyne incognita

This investigation was undertaken to determine the effect of culture filtrates of different isolates of Fusarium oxysporum f. sp. lycopersici on mortality of Meloidogyne incognita juveniles and egg hatching. It was observed that different concentrations including standard extract (S.E), 1:10 and 1:100 dilutions of all fungal filtrates inhibited egg hatch when compared with control. Minimum mortality and maximum hatching was observed in BRT (showing least mortality) isolate of F. oxysporum, while maximum mortality and minimum egg hatching was recorded in BGT (showing maximum mortality) isolate. Larval mortality was decreased with a decrease in concentration and the least mortality was observed in 1:100 when compared with SE and 1:10. The potentiality of both the isolates (BRT and BGT) against root-knot nematode M. incognita was confirmed by the pathogenicity test on tomato. These observations confirmed that F. oxysporumisolates possesses variability in pathogenicity ranging from pathogenic to bio-control agent. The plants inoculated with BRT isolate failed to show wilt symptoms while plants inoculated with BGT isolate showed wilt indices.     

Potentiality of different isolates of wilt fungus Fusarium oxysporum collected from rhizosphere of tomato against root-knot nematode Meloidogyne incognita

This investigation was undertaken to determine the effect of culture filtrates of different isolates of Fusarium oxysporum f. sp. lycopersici on mortality of Meloidogyne incognita juveniles and egg hatching. It was observed that different concentrations including standard extract (S.E), 1:10 and 1:100 dilutions of all fungal filtrates inhibited egg hatch when compared with control. Minimum mortality and maximum hatching was observed in BRT (showing least mortality) isolate of F. oxysporum, while maximum mortality and minimum egg hatching was recorded in BGT (showing maximum mortality) isolate. Larval mortality was decreased with a decrease in concentration and the least mortality was observed in 1:100 when compared with SE and 1:10. The potentiality of both the isolates (BRT and BGT) against root-knot nematode M. incognita was confirmed by the pathogenicity test on tomato. These observations confirmed that F. oxysporumisolates possesses variability in pathogenicity ranging from pathogenic to bio-control agent. The plants inoculated with BRT isolate failed to show wilt symptoms while plants inoculated with BGT isolate showed wilt indices.     

Degradation pathways of low-ethoxylated nonylphenols by isolated bacteria using an improved method

Nonylphenol ethoxylates (NPEOs) with low ethoxylation degree (NP_av2EO; containing two ethoxy units on average) and estrogenic properties are the intermediate products of nonionic surfactant NPEOs. To better understand the environmental fate of low-ethoxylated NPEOs, phylogenetically diverse low-ethoxylated NPEO-degrading bacteria were isolated from activated sludge using gellan gum as the gelling reagent. Four isolates belonging to four genera, i.e., Pseudomonas sp. NP522b in γ-Proteobacteria, Variovorax sp. NP427b and Ralstonia sp. NP47a in β-Proteobacteria, and Sphingomonas sp. NP42a in α-Proteobacteria were acquired. Ralstonia sp. NP47a or Sphingomonas sp. NP42a, have not been reported for the degradation of low-ethoxylated NPEOs previously. The biotransformation pathways of these isolates were investigated. The first three strains (NP522b, NP427b, and NP47a) exhibited high NP_av2EO oxidation ability by oxidizing the polyethoxy (EO) chain to form low-ethoxylated nonylphenoxy carboxylates, and then further oxidizing the alkyl chain to form carboxyalkylphenol polyethoxycarboxylates. Furthermore, Sphingomonas sp. NP42a degraded NP_av2EO through a nonoxidative pathway with nonylphenol monoethoxylate as the dominant product.     

Mycobiota and mycotoxins in bee pollen collected from different areas of Slovakia

Contamination by microscopic fungi and mycotoxins in different bee pollen samples, which were stored under three different ways of storing as freezing, drying and UV radiation, was investigated. During spring 2009, 45 samples of bee-collected pollen were gathered from beekeepers who placed their bee colonies on monocultures of sunflower, rape and poppy fields within their flying distance. Bee pollen was collected from bees' legs by special devices placed at the entrance to hives. Samples were examined for the concentration and identification of microscopic fungi able to grow on Malt and Czapek-Dox agar and mycotoxins content [deoxynivalenol (DON), T-2 toxin (T-2), zearalenone (ZON) and total aflatoxins (AFL), fumonisins (FUM), ochratoxins (OTA)] by direct competitive enzyme-linked immunosorbent assays (ELISA). The total number of microscopic fungi in this study ranged from 2.98 ± 0.02 in frozen sunflower bee pollen to 4.06 ± 0.10 log cfu.g(-1) in sunflower bee pollen after UV radiation. In this study, 449 isolates belonging to 21 fungal species representing 9 genera were found in 45 samples of bee pollen. The total isolates were detected in frozen poppy pollen 29, rape pollen 40, sunflower pollen 80, in dried poppy pollen 12, rape pollen 36, sunflower 78, in poppy pollen after UV radiation treatment 54, rape 59 and sunflower 58. The most frequent isolates of microscopic fungi found in bee pollen samples of all prevalent species were Mucor mucedo (49 isolates), Alternaria alternata (40 isolates), Mucor hiemalis (40 isolates), Aspergillus fumigatus (33 isolates) and Cladosporium cladosporioides (31 isolates). The most frequently found isolates were detected in sunflower bee pollen frozen (80 isolates) and the lowest number of isolates was observed in poppy bee pollen dried (12 isolates). The most prevalent mycotoxin of poppy bee pollen was ZON (361.55 ± 0.26 μg.kg(-1)), in rape bee pollen T-2 toxin (265.40 ± 0.18 μg.kg(-1)) and in sunflower bee pollen T-2 toxin (364.72 ± 0.13 μg.kg(-1)) in all cases in frozen samples.     

Extensive biodegradation of polychlorinated biphenyls in Aroclor 1242 and electrical transformer fluid (Askarel) by natural strains of microorganisms indigenous to contaminated African systems

Evidence for substantial aerobic degradation of Aroclor 1242 and Askarel fluid by newly characterized bacterial strains belonging to the Enterobacter, Ralstonia and Pseudomonas genera is presented. The organisms exhibited degradative activity in terms of total PCB/Askarel degradation, degradation of individual congeners and diversity of congeners attacked. Maximal degradation by the various isolates of Askarel ranged from 69% to 86% whereas, Aroclor 1242, with the exception of Ralstonia sp. SA-4 (9.7%), was degraded by 37% to 91%. PCB analysis showed that at least 45 of the representative congeners in Aroclor 1242 were extensively transformed by benzoate-grown cells without the need for biphenyl as an inducer of the upper degradation pathway. In incubations with Aroclor 1242, no clear correlation was observed between percentage of congener transformed and the degree of chlorination, regardless of the presence or absence of biphenyl. Recovery of significant but nonstoichiometric amounts of chloride from the culture media showed partial dechlorination of congeners and suggested production of partial degradation products. Addition of biphenyl evidently enhanced dechlorination of the mixture by some isolates. With the exception of Ralstonia sp. SA-5, chloride released ranged from 24% to 60% in the presence of biphenyl versus 0.35% to 15% without biphenyl.     

Characterization of phenol degradation by high-efficiency binary mixed culture

Background, aim, and scope  

Two new high phenol-degrading strains, Micrococcus sp. and Alcaligenes faecalis JH 1013, were isolated. The two isolates could grow aerobically in mineral salts medium containing phenol as a sole carbon source at concentration of 3,000 mg L⁻¹. It was found that the binary mixed culture of the two isolates possessed good potential for phenol removal.     

Occurrence of benzotriazoles in the rivers Main,Hengstbach, and Hegbach (Germany)

Background, aim, and scope  

Benzotriazoles (BT) as 1H-benzotriazole (1H-BT), 5-methyl-1H-benzotriazole (5Me-BT), and 4-methyl-1H-benzotriazole (4Me-BT) are frequently used as corrosion inhibitors in dish washer detergents, aircraft de-icing/anti-icing fluids (ADAF), automotive antifreeze formulations, brake fluids, fluids for industrial cooling systems, metal-cutting fluids, and in solid cooling lubricants. Discharge of treated municipal waste water and controlled over-runs of combined waste water sewers are potential point sources for BT in rivers. The aim of this monitoring study was to yield an overview on exposure concentrations and loads of BT in the German rivers Main, Hengstbach, and Hegbach.     

Metabolism of chlorinated biphenyls: use of 3,3'- and 3,5-dichlorobiphenyl as sole sources of carbon by natural species of Ralstonia and Pseudomonas

Ralstonia sp. SA-3, Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 are natural strains with a novel capacity to utilize meta-substituted dichlorobiphenyls (diCBs) hitherto not known to serve as a sole source of carbon and energy for polychlorobiphenyl-degraders. In growth experiments, axenic cultures of isolates grew logarithmically on 3,3'-diCB with generation times that ranged insignificantly (t-test, P>0.05) from 30.4 to 33.8 h. Both 3-chlorobenzoate (3-CBA) and chloride produced as metabolites were recovered in non-stoichiometric quantities. The release of chloride by the cultures lagged substantially, indicating that the initial dioxygenase attack preceded cleavage of carbon-chloride bonds and that chloride must have been released from the chlorinated hydroxypentadienoate. In the case of 3,5-diCB, SA-3 and SA-6 metabolised this substrate primarily to 3,5-CBA. The lack of chloride in the culture media coupled with stoichiometric recovery of 3,5-CBA suggests that growth by these strains occurred predominantly at the expense of the unsubstituted phenyl ring. The unique metabolic properties of these three aerobic isolates point to their potential usefulness as seeds for bioremediation of PCBs polluted environments without the need for repeated inoculation or supplementation by a primary growth substrate such as biphenyl.