Early succession of bacterial biofilms in paper machines

Formation of biofilms causes severe problems in paper machines, and hence financial costs. It would be preferable to prevent attachment of the primary-colonizing bacteria than to control the growth of secondary communities, which are sheltered by exopolysaccharide slime layers. We have therefore investigated the early succession of paper-machine biofilms by incubating stainless-steel test coupons in the process water-flow lines in two paper machines operating in slightly alkaline conditions in temperatures (45 and 49°C) supporting thermophilic microbes. Microbial succession was profiled using length heterogeneity analysis of PCR-amplified 16S rRNA genes (LH-PCR) and linking the sequence data of the created 16S rRNA gene libraries to the dominant LH-PCR peaks. Although the bacterial fingerprints obtained from the attached surface communities varied slightly in different samples, the biomarker signals of the dominating primary-colonizing bacterial groups remained high over time in each paper machine. Most of the 16S rRNA gene copies in the early biofilms were assigned to the genera Rhodobacter, Tepidimonas, and Cloacibacterium. The dominance of these sequence types decreased in the developing biofilms. Finally, as phylogenetically identical primary-colonizers were detected in the two different paper mills, the machines evidently had similar environmental conditions for bacterial growth and potentially a common source of contamination.     

Isolation and characterization of nickel-resistant microflora from serpentine soils of Andaman

Serpentine soils of Andaman Islands, India characteristically contain high levels of nickel, cobalt and chromium and are colonized by indigenous nickel-hyperaccumulating plants. Attempts have been made to isolate and characterize nickel-resistant microorganisms from these hitherto unexplored naturally nickel-percolated soils. The majority of the nickel-resistant organisms showed a minimum inhibitory concentration (MIC) of Ni²⁺ ranging from 300 to 400 mg/l and about 3.4% of the total 89 isolates representing bacterial strains were able to grow at 400 mg/l Ni²⁺. The potent Ni²⁺-resistant strains AND305 and AND603 were tentatively identified as Pseudomonas spp. and strain AND408 as Bacillus sp. following detailed analysis of morphological and physio-biochemical characteristics. Growth kinetics of these Ni²⁺-resistant bacteria showed a prolonged lag phase in Ni²⁺-containing media, which extended with increasing nickel concentration. In addition to Ni²⁺, these isolates were also resistant to Co²⁺, Cd²⁺, Cr⁶⁺, Fe³⁺, Cu²⁺, Mg²⁺, Mn²⁺(50–200 mg/l) and Hg²⁺ (0.5–2.0 mg/l) and the multiple metal-resistance of the isolates were also associated with the resistance to antibiotics ampicillin, cycloserine and penicillin G.     

Production,purification and characterization of pectinase from a Bacillus sp. DT7

A soil isolate, Bacillus sp. DT7 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel filtration and ion exchange chromatography, this enzyme was purified and found to have a molecular mass of 106 kDa. The purified enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl₂ and mercaptoethanol significantly enhanced pectinase activity of the purified enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

Occurrence and characterization of actinobacteria and thermoactinomycetes isolated from pulp and board samples containing recycled fibres

The aim of this study was to characterize the actinobacterial population present in pulps and boards containing recycled fibres. A total of 107 isolates was identified on the basis of their pigmentation, morphological properties, fatty acid profiles and growth temperature. Of the wet pulp and water sample isolates (n=87), 74.7% belonged to the genus Streptomyces, 17.2% to Nocardiopsis and 8.0% to thermoactinomycetes, whereas all the board sample isolates (n=20) were thermoactinomycetes. The identification of 53 isolates was continued by molecular methods. Partial 16S rDNA sequencing and automated ribotyping divided the Streptomyces isolates (n=31) into 14 different taxa. The most common streptomycetes were the mesophilic S. albidoflavus and moderately thermophilic S. thermocarboxydus. The Nocardiopsis isolates (n=11) belonged to six different taxa, whereas the thermoactinomycetes were mainly members of the species Laceyella sacchari (formerly Thermoactinomyces sacchari). The results indicated the probable presence of one or more new species within each of these genera. Obviously, the drying stage used in the board making processes had eliminated all members of the species Streptomyces and Nocardiopsis present in the wet recycled fibre pulp samples. Only the thermotolerant endospores of L. sacchari were still present in the final products. The potential of automated ribotyping for identifying actinobacteria was indicated, as soon as comprehensive identification libraries became available.     

Hydrolysis of wheat bran,rice bran and jute powder by immobilized enzymes from Macrophomina phaseolina

The stability of cellulolytic and hemicellulolytic enzymes from Macrophomina phaseolina improved on immobilization and was 1.5 to 2-fold more active against pre-treated wheat bran, rice bran or jute powder. The hydrolysis efficiency of the catalyst increased with a decrease in its particle size. About 80% (w/v) of the sugar obtained from wheat bran was assimilated by Saccharomyces sp., whereas the corresponding values for rice bran and jute powder were about 70 and 50% (w/v), respectively.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.     

花生种子相关促生菌分离鉴定及功能评价

【背景】利用微生物促进植物健康生长是农业可持续发展的重要方向之一,而种子相关的促生菌可在植物生命周期早期与植物相互作用,对植物健康生长具有重要意义。【目的】发掘与利用种子相关促生菌的前提是筛选获得促生菌菌种资源,验证其益生能力,为其进一步应用与机理研究提供依据与支持。【方法】以花生种子为研究对象,从种子表面及种子内部分离纯化多株菌,测定菌株的固氮、解磷、解钾、吲哚乙酸合成和铁载体合成等促生能力,并验证菌株对常见植物病原菌的生长抑制特性;通过16S rRNA基因序列进行系统发育分析,确定分类地位;通过生物膜形成能力及根际定殖能力测定菌株在植物根际的生存能力;最后通过催芽及盆栽试验测定菌株对花生种子发芽及幼苗生长的影响。【结果】从花生种子表面、种子内和胚根内分离筛选到41株菌,均有吲哚乙酸合成能力,其中35株有固氮能力,2株有铁载体分泌能力,14株有植物病原菌生长抑制能力。各选一株为代表的菌株,即PS3、PE5和PR5,经16S rRNA基因序列比对分析鉴定为芽孢杆菌属(Bacillus)。PS3、PE5和PR5均可在MSgg液体培养基表面形成褶皱较强的生物膜,也可在花生根际形成有效定殖。催芽试验结果表明经过促生菌浸种后花生种子萌发率明显提高,在第2天时,PS5将发芽率由14.17%提高至38.33%,PE5发芽率提高至30.83%,PR5发芽率提高至39.17%。三株菌能够明显促进花生幼苗生长,PS5对花生幼苗苗高、根长、鲜重和干重分别提高21.82%、22.20%、37.11%和35.64%,PE5分别提高17.45%、18.93%、26.10%和21.18%,PR5分别提高23.11%、23.92%、38.66%和37.47%。【结论】筛选获得的花生种子相关促生菌,具有促进植物生长的潜力,明显促进种子萌发及幼苗生长,是良好的促生菌生物资源,具有较好的应用潜力。     

芽胞杆菌产生的脂肽类抗生素的结构和应用

脂肽类抗生素不仅对细菌、真菌有抗菌作用,而且对支原体、寄生虫和病毒等也具有显著的抑制作用。其主要包括表面活性素(Surfactins)、伊枯草菌素(Iturins)和芬芥素(Fengycins),其中Iturins和Fengycins有抗真菌活性,Surfactins有抗支原体、抗病毒等活性。综述了芽胞杆菌产生的脂肽类抗生素的结构、功能和应用,为其在农业和医药的研究与应用提供参考。     

水貂源高产蛋白酶枯草芽孢杆菌的分离筛选及酶学性质

【背景】开发、筛选优良益生菌菌种是当下畜牧业的研究热点,益生菌的潜在功能也被广为挖掘。【目的】分离、筛选具有良好耐受性且高产胞外蛋白酶的菌株,研究其生物学特性和酶学性质,为后续微生物蛋白酶的制备和微生态制剂的开发提供菌种资源。【方法】采集健康水貂新鲜粪便,配制酪蛋白培养基初筛和优化Folin-酚法复筛,对筛选菌进行生物学特性研究,获得产蛋白酶能力较强、耐受性较优的菌株,并进行常规鉴定和分子生物学鉴定,最后对蛋白酶酶学性质进行研究。【结果】筛选得到一株高产蛋白酶、耐受性较优的芽孢杆菌,经鉴定为枯草芽孢杆菌(Bacillussubtilis),编号为3。在初始发酵培养基条件下,酶学性质研究结果表明,该蛋白酶的最适反应温度为70℃,最适反应pH值为9.0,最佳金属离子激活剂为K+,Cu2+和Fe2+对酶活力有明显的抑制作用,在20%浓度的有机溶剂作用时蛋白酶未变性失活。【结论】从水貂粪便中分离获得一株具有良好的生物学特性、酶学性质和碱性蛋白酶活性的枯草芽孢杆菌,为该菌株在实际生产应用中提供了基础保障。     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

分离自苏云金芽胞杆菌的首例细菌乌头酸异构酶TbrA的酶学性质研究(英文)

【目的】乌头酸异构酶(aconitate isomerase,AI)可介导具有多重生物学活性及应用潜力的小分子物质反式乌头酸(trans-aconiticacid,TAA)的合成。本文通过表征来自苏云金芽胞杆菌的生物体首条AI基因(tbrA)的产物——TbrA蛋白的催化性质,填补人们对于AI酶学特性的认识。【方法】我们利用大肠杆菌Rosetta菌株和Ni²⁺柱亲和纯化获得了His6-TbrA蛋白,并在体外通过HPLC检测了产物生成及对应酶活。【结果】TbrA蛋白的最适pH、温度与离子强度分别为8.0,37°C和25 mmol/L。TbrA在10°C时仍保留约60%的活性,展现了较好的耐低温特性。金属离子Mg²⁺、Ca²⁺与还原剂DTT可显著增强TbrA活性,而Fe²⁺、Cu²⁺、Zn²⁺、Mn²⁺则强烈抑制TbrA活性。TbrA将顺式乌头酸(cis-aconitic acid,CAA)异构为TAA的正反应K_m、V...     

Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01

We isolated the feather-degrading Bacillus pseudofirmus FA30-01 from the soil sample of poultry farm. The isolate completely degraded feather pieces after liquid culture at 30°C (pH 10.5) for 3 days. Strain FA30-01 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. pseudofirmus based on 16S rDNA analysis. The keratinase enzyme produced by strain FA30-01 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The refinement level was 14.5-fold. The molecular weight of this enzyme was 27.5 kDa and it had an isoelectric point of 5.9. The enzyme exhibited activity at pH 5.1–11.5 and 30–80°C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 8.8–10.3 and 60°C, respectively. This enzyme is one of the serine-type proteases. Subtilisin ALP I and this enzyme had 90% homology in the N-terminal amino acid sequence. Since this enzyme differed from ALP I in molecular weight, heat resistance and isoelectric point, they are suggested to be different enzymes.     

Production of mycelial protein and hydrolytic enzymes from paper mill sludges by cellulolytic fungi

Summary Characterization of lignocellulosic wastes from three paper mills in New York State indicated that a kraft mill sludge contained substantial quantities of utilizable cellulose and hemicellulose. This residue was tested as a carbon source for seven cellulolytic fungi.Trichoderma reesei DAOM 167654 accumulated a product of over 22% crude protein, and caused a conversion of sludge to protein of almost 15% in 3 days growth in shake flasks.T. reesei also produced the highest levels of cellulase, whileT. longibrachiatum produced more xylanase (35 units/ml) than other fungi examined.     

Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

Summary The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C₂). The purified lipase showed no protease or thioesterase activity.     

Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species

The purpose of this study was to investigate the production of various enzymes by oral and non-oral black-pigmented Bacteroides species using chromogenic substrates. The 19 substrates present in the API ZYM system did not differentiate between B. melaninogenicus, B. denticola, B. loescheii and B. levii. The asaccharolytic black-pigmented Bacteroides species showed each species specific enzyme activity, however, differences were based on one enzyme only as far as B. asaccharolyticus and B. endodontalis are concerned. An extended number of 40 chromogenic substrates were tested in order to find more species specific enzyme. With a set of 20 substrates it appeared to be possible to discriminate between all species tested. The possibility to use enzymes for the identification of black-pigmented Bacteroides is discussed.     

Effects of some groundnut packaging methods and protection with Ocimum and Syzygium powders on kernel infection by fungi

Powders from the leaves of Ocimum gratissimum and cloves of Syzygium aromaticum were used as protectants at 3% (w/w) in combination with various packaging methods to store 3.5 kg groundnut kernel samples (9.3% moisture) artificially inoculated with Aspergillus parasiticus. Phostoxin-protected and unprotected samples were the controls. Packaging was accomplished with (i) Jute bags; JB (ii) Interlaced polypropylene bags; IPPB (iii) Polyethylene bags; PB (iv) PB inserted into IPPB and (v) PB inserted into JB. Selected treatments were repeated concurrently with naturally infected kernels (6.6% moisture). With 9.3% moisture kernels, there was a highly significant protectant, packaging method, and protectant X packaging method effect on protection of kernels from fungal infection at 2, 4, and 6 months. Packaging with JB and IPPB with or without plant powders gave 100% protection against fungi but insect infestation was prevented only when the Syzygium powder was used. When PB was used either singly or in combination with JB and IPPB, 100% protection from fungi was achieved up to 2 months with the Ocimum and up to 4 months with the Syzygium powder. The phostoxin treatment also gave 100% protection with JB and IPPB packaging but was ineffective with PB packaging. Kernels packaged with PB without the powders were extensively mouldy. Kernels with natural mycoflora (6.6% moisture) were free from fungi at 6 months regardless of the protectant and packaging used. In further tests, the Syzygium powder, at 3% and in combination with JB-packaging, effectively suppressed cross infection of healthy kernels (12% moisture) by fungi from diseased kernels when both kernel types occurred in the same lot. At 18.5% kernel moisture and with identical packaging, the Syzygium powder at 3%, was not as effective. This revised version was published online in June 2006 with corrections to the Cover Date.     

椰子织蛾幼虫肠道细菌的初步分离鉴定及功能分析

[目的] 研究椰子织蛾幼虫肠道微生物的种类和功能,以揭示其消化利用寄主老叶的机制。[方法] 采用传统微生物分离培养技术分离培养肠道细菌,用16S rRNA基因序列分析的方法鉴定菌株,采用透明圈染色法对所得菌株进行功能性验证。[结果] 基因序列检测对比鉴定得到9种可培养细菌菌株,主要属于变形菌门和厚壁菌门以及放线菌门;功能性验证结果表明,伯克霍尔德氏菌、解淀粉芽孢杆菌、贝莱斯芽孢杆菌、蜡样芽孢杆菌菌株具有纤维素降解酶,寒气玫瑰单胞菌、解淀粉芽孢杆菌含木聚糖降解酶。[结论] 椰子织蛾肠道中存在可培养的具有降解纤维素及木聚糖能力的细菌,这些细菌可能有助于椰子织蛾取食消化椰子等老叶,研究所获得的肠道微生物菌株也为后续研究该虫与环境的关系及相关菌株应用于农业、能源、环保价值的探索提供帮助。     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.