Selection and characterization of feather-degrading bacteria from canola meal compost

Canola meal that contains a high level of protein (40% crude protein) was used as compost material for the isolation of feather-degrading bacteria. After 7 and 14 days, bacteria were isolated from compost amended and unamended with soil. Eighty bacterial isolates from canola meal compost were then grown on milk-agar and isolates that produced proteolytic enzymes were identified by the formation of clear haloes around the colonies. A feather medium was chosen for a secondary selection of feather-degrading isolates. Of the eight isolates that hydrolyzed milk protein, five isolates hydrolyzed feathers. Their keratinolytic activities were subsequently confirmed by an assay using azo-keratin as substrate. Seven of the eight bacteria that hydrolyzed milk protein were Bacillus spp, and all five isolates that hydrolyzed feathers were strains of Bacillus licheniformis. Protease inhibition studies indicated that serine proteases are the predominant proteolytic enzymes produced by these feather-degrading isolates. Received 02 April 1999/ Accepted in revised form 17 June 1999     

Soil bacterial strains able to grow on the surface of oxidized polyethylene film containing prooxidant additives

Twelve bacterial strains able to adsorb and grow on the surface of oxidized low-density polyethylene film containing prooxidant additives were isolated from three forest soils and subsequently identified. Most of them belonged to different genera of the proteobacteria group; however, three of the isolates were Rhodococcus strains. With the exception of one of the Rhodococcus strains, the isolates did not exhibit significant hydrophobicity of their cell surfaces. The study showed that bacteria capable of adhering to the surface of oxidized polyethylene, growing there and possibly biodegrading its oxidation products are not rare in forest soils and that they belong to different taxonomical groups common in soil environment.     

Isolation, Identification, and Characterization of a Feather-Degrading Bacterium

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10⁹ cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10⁷ cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.     

Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia

Root-associated bacteria can have beneficial effects on their host plants. Microbial products can promote and stimulate plant growth or lead to bioprotection against pathogens. This study aimed to isolate putatively beneficial bacteria from traditional cereals grown by subsistence farmers in the Kavango of Namibia. Bacteria were isolated from surface-sterilized roots of Pennisetum glaucum, Sorghum bicolor, and Zea mays, and subjected to phenotypic and phylogenetic analyses. A total of 44 root-associated bacterial strains were isolated. From 33 distinct isolates, 22 belonged to Firmicutes and Actinobacteria, while 11 were Proteobacteria. Eleven novel phylotypes were among the isolates. Features known to contribute to plant growth-promotion and biocontrol were tested in vitro and revealed promising candidates with multiple beneficial characteristics. This is the first report on the characterization of native isolates associated with important agriculture crops in the Kavango region of Namibia. Such isolates have the potential for application as inoculants adapted to poor soils and local crops. Desiccation-tolerant or sporulating Gram-positive bacteria are of particular interest for this region, which is characterized by a long dry season.     

Assessment of Keratinase and Other Hydrolytic Enzymes in Thermophilic Bacteria Isolated from Geothermal Areas in Patagonia Argentina

Thermophilic aerobic bacteria were isolated from two geothermal areas in Neuquén province using two different enrichment methods and a total of 30 isolates were obtained. From chicken feather enrichment cultures, strains affiliated to Bacillus cytotoxicus and Bacillus licheniformis were isolated and all of them demonstrated the capability to degrade completely chicken feather. A preliminary research on biotechnological enzymes' potential demonstrated that all the isolates displayed at least one of the extracellular hydrolytic enzymes tested. Most of the isolates showed protease, inulinase and/or pectinase activities, while cellulase and xylanase activities were less common. In light of these findings, geothermal areas of Argentina may be considered as a potential source of thermophilic bacteria able to produce many industrially relevant enzymes.     

Aerobic Microbial Degradation of Glucoisosaccharinic Acid

α-Glucoisosaccharinic acid (GISA), a major by-product of kraft paper manufacture, was synthesized from lactose and used as the carbon source for microbial media. Ten strains of aerobic bacteria capable of growth on GISA were isolated from kraft pulp mill environments. The highest growth yields were obtained with Ancylobacter spp. at pH 7.2 to 9.5. GISA was completely degraded by cultures of an Ancylobacter isolate. Ancylobacter cell suspensions consumed oxygen and produced carbon dioxide in response to GISA addition. A total of 22 laboratory strains of bacteria were tested, and none was capable of growth on GISA. GISA-degrading isolates were not found in forest soils.     

Chemical warfare? Effects of uropygial oil on feather‐degrading bacteria

Anti-microbial activity is a commonly suggested but rarely tested property of avian uropygial oil. Birds may defend themselves against feather-degrading and other potentially harmful bacteria using this oil. We preliminarily identified 13 bacterial isolates taken from the plumage of wild house finches Carpodacus mexicanus , measured bacterial production of the enzyme keratinase as an index of feather-degrading activity, and used the disc-diffusion method to test bacterial response to uropygial oil of house finches. For comparison, we performed the same tests on a type strain of the known feather-degrading bacterium Bacillus licheniformis . Uropygial oil inhibited the growth of three strongly feather-degrading isolates (including Bacillus licheniformis ), as well as one weakly feather-degrading isolate and one non-feather-degrading isolate. Uropygial oil appeared to enhance the growth of one weakly feather-degrading isolate. Growth of the remaining isolates was unaffected by uropygial oil. These results suggest that birds may defend themselves against some feather-degrading bacteria using uropygial oil.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

MALDI-TOF-MS and 16S rRNA characterization of lead tolerant metallophile bacteria isolated from saffron soils of Kashmir for their sequestration potential

Toxic metal contamination in soils due industrialization is nowadays a concern to the scientists worldwide. The current study deals with the evaluation of response and tolerance by isolated metallophilic bacteria in different lead concentrations (100 ppm to 1000 ppm). By taking optical densities of the isolates, the minimum inhibitory concentration (MIC) of Pb²⁺ were determined.16S rRNA and MALDI-TOF MS were used for the identification of the bacteria. Total of 37 isolates were observed, among them 04 (Staphylococcus equorum, Staphylococcus warneri, Bacillus safensis and Bacillus thuringiensis), isolated were detected having efficacy of Pb²⁺tolerance and sequestration at varying MIC. Furthermore, B. thuringiensis was observed to have highest (900 ppm) tolerance for lead and lowest (500 ppm) for Staphylococcus warneri. Moreover, the highest (65.3%) sequestration potential has been observed for B. thuringiensis and least (52.8%) for S. warneri. The tolerance and sequestration potential properties of these isolated species can be utilised to exterminate heavy metals and reduce their toxicity from the contaminated environment.     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.     

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45–55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.     

Alkalitolerant micromycetes in acidic and neutral soils of the temperate zone

A wide diversity of micromycetes from various taxonomic groups in acidic and neutral soils is known from the literature data. In the present work, the fungi isolated from these soils and capable of growth at high pH are analyzed. The fungi were isolated from acidic sod-podzol and neutral cultivated soils by plating on alkaline agar (pH 10.0–10.5). Their identification was carried out using morphological, cultural, and molecular genetic criteria. Phylogenetic analysis was performed and the rates of linear growth within a broad pH range (4.0–10.4) were determined. The isolates represented a polyphyletic group of ascomycetes (Sordariomycetes), which included members of Plectosphaerellaceae (5 species) and various families of Hypocreales (4 species). The most common species were Gibellulopsis nigrescens, Acrostalagmus luteoalbus, Chordomyces antarcticum, and Plectosphaerella spp. Investigation of fungal growth at different pH values revealed all isolates to be alkalitolerant, with no alkaliphilic fungi isolated from acidic sod-podzol and neutral cultivated soils. Although the group of isolates was polyphyletic and its members originated from different ecological and trophic niches, most alkalitolerant isolates exhibited common morphological traits with acremonium- and verticillium-like conidial spore formation, abundant slime formation, and a tendency for aggregation of their mycelium in bundles. Our research confirmed the presence of fungi with alkalitolerant adaptation to external pH in the sod-podzolic and cultivated soils of the Moscow region.     

Nonsymbiotic nitrogen-fixing micro-organisms in forest and tundra soils

Summary Soil samples obtained from various forested sites in North Carolina and Washington and from Alaskan tundra were examined for the presence of heterotrophic, nonsymbiotic nitrogen-fixing micro-organisms. Aerobic, nitrogen-fixing micro-organisms were not isolated from any of the soils examined. Estimates of anaerobic nitrogen-fixing bacteria in these soils ranged from 50,000 to 2,000,000/g when a dilution plate technique and a medium supplemented with potato extract was used. However, the isolation of individual colonies from the dilution plates showed that many of these bacteria were unable to fix nitrogen. Soil populations well below 100,000/g were generally indicated by this colony isolation technique. Differentiation of the colonies by size improved the accuracy of the dilution plate estimates somewhat. Dilution tube procedures appeared more suitable for obtaining accurate counts of nitrogen-fixing anaerobes in the soil than the use of dilution plates. The predominant nitrogen-fixing bacterium in most soils was a facultative anaerobe,Bacillus polymyxa. Appreciable numbers of nitrogen-fixing clostridia were also found in several tree nursery soils but were seldom isolated from forest and tundra samples. The clostridia isolated were classified asClostridium butyricum andC. pasteurianum. Variations in the fermentation patterns of these bacteria occurred when the nitrogen supply of the medium was altered. TheC. butyricum isolates were all from forest soils while all except one of theC. pasteuranium isolates were from tundra soils. Paper number2998 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C     

Feather keratin hydrolysis by a Vibrio sp. strain kr2

The aim of the study was to characterize feather-degrading bacteria isolated from poultry industry waste. A Vibrio sp. strain kr2 producing a high keratinolytic activity when cultured on native feather-containing broth was isolated. The bacterium grew with an optimum at pH 6.0 and 30 degrees C, where maximum featherdegrading activity was also observed. Keratinase production was similar at both 25 and 30 degrees C, while the maximum concentration of soluble protein was reached at 30 degrees C. Reduction of disulphide bridges was also observed, increasing with cultivation time. The keratinase of strain kr2 was active on azokeratin, azocasein, benzoyl-arginine-p-nitroanilide and Ala-Ala-p-nitroanilide as substrates. The amino acid composition of the feather hydrolysate was determined, presenting similarities with that reported for feather lysate, feather meal and raw feathers. A novel feather-degrading bacterium was isolated and characterized, showing high keratinolytic activity. Complete feather degradation was achieved during cultivation. Strain kr2 shows potential for use for biotechnological processes involving keratin hydrolysis.     

Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.The toxicity of phenolic compounds in the environment has fostered studies of bacteria that are able to tolerate and/or metabolize high levels of these compounds, particularly under anaerobic conditions (1, 4, 14, 21, 30, 36). Tannins are secondary polyphenolic compounds known primarily for their ability to bind to and precipitate proteins and other macromolecules. Tannins have been found in many habitats, including sewage sludge, forest litter, and the rumen (9, 14, 15, 28). Bacteria capable of degrading or tolerating tannins have been isolated from sewage sludge (14) and from the alimentary tracts of koalas (Phascolarctos cinereus) (33), goats (Capra hircus) (4, 30), and horses (Equus caballus) (31). Most of the isolates have been characterized phenotypically, and phylogenetic characterization has been limited to studies conducted in Australia (4, 34, 35) and Japan (31). Little is known about the geographic diversity and host species diversity of tannin-tolerant and tannin-degrading bacteria.The objective of this study was to characterize six recently isolated tannin-tolerant bacteria by examining their phenotypic characteristics and molecular phylogeny. These bacteria were isolated from the ruminal contents of goats (C. hircus), sheep (Ovis aries), white-tail deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni), all of which had consumed forage containing tannins. Our goal was to genetically and biochemically characterize tannin-tolerant bacteria isolated from different host animals in various geographic locations.     

Isolation and identification of threeCellulomonas spp. from forest soils

Three stains of cellulose-degrading, aerobic, mesophilic bacteria were isolated from forest soils and, from their cultural, biochemical, and physiological characteristics, they were identified as members of the genusCellulomonas. Unusual biochemical characteristics, e.g. urea hydrolysis, were observed in two isolates. These characteristics have not previously been reported for cellulomonads and may prove to be significant for characterization ofCellulomonas spp. The isolates were able to use urea as a N source in cellulose fermentation. All three strains were motile, with one to four peritrichous flagella observed. Amino acid and polysaccharide composition of the cell walls of the three isolates were identical.     

Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes

A novel feather-degrading bacterium named CA-1 was isolated from the gut of the spider Chilobrachys guangxiensis, which degrades native whole chicken feathers within 20 h. The CA-1 was confirmed to belong to Stenotrophomonas maltophilia based on morphologic and molecular analysis. Maximum feather degradation activity of the bacterium was observed at 37 °C in basal feather medium (NaCl 0.5 g/L, KH₂PO₄ 0.3 g/L, K₂HPO₄ 0.4 g/L, feather powder 10.0 g/L, pH 8.0), which was inhibited when glucose and ammonium nitrate were added in the medium. Furthermore, the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC–MS/MS. Three enzymes, namely alkaline serine protease (29.1 kDa), ABC transporter permease (27.5 kDa), and alkaline phosphatase (40.8 kDa), were isolated and identified from the supernatant of the optimal culture and were considered to play principal roles. On the other hand, the potential synergic effects of the three proteins in S. maltophilia CA-1 feather degradation system were analyzed theoretically. CA-1 may product outer-membrane vesicles comprised of membranes and periplasmic proteins in the feather medium. The newly identified CA-1 and its synergic enzymes provide a new insight into further understanding the molecular mechanism of feather degradation by microbes. They also have potential application in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the three newly identified enzymes.     

一株羽毛降解菌的分离与鉴定

【目的】从土壤中分离并鉴定羽毛降解菌,测定其生长最适温度及起始pH,并观察酶活动态。【方法】采用系列稀释法和选择培养基法筛选目的菌株,基于16S rRNA基因序列及Biolog方法鉴定其分类地位,利用全自动生长曲线分析仪监测菌株的最适生长条件,并通过测定蛋白水解活性观察其酶活动态。【结果】从混合羽毛的土壤样品中筛选到一株羽毛降解菌,命名为菌株GIMN1.015,初步判定该菌株属于芽孢八叠球菌属(Sporosarcina)。最适生长pH为9.0,温度为30°C。蛋白水解活性最高值出现在培养后96 h。【结论】菌株GIMN1.015在利用羽毛角蛋白资源中具有潜在的应用价值。这是芽孢八叠球菌在羽毛降解方面的首次报道。     

Plant growth-promoting activities of fluorescent pseudomonads,isolated from the Iranian soils

Fluorescent pseudomonads are among the most influencing plant growth-promoting rhizobacteria in plants rhizosphere. In this research work the plant growth-promoting activities of 40 different strains of Pseudomonas fluorescens and Pseudomonas putida, previously isolated from the rhizosphere of wheat (Triticum aestivum L.) and canola (Brassica napus L.) and maintained in the microbial collection of Soil and Water Research Institute, Tehran, Iran, were evaluated. The ability of bacteria to produce auxin and siderophores and utilizing P sources with little solubility was determined. Four strains of Wp1 (P. putida), Cfp10 (Pseudomonas sp.), Wp150 (P. putida), and Wp159 (P. putida) were able to grow in the DF medium with ACC. Thirty percent of bacterial isolates from canola rhizosphere and 33% of bacterial isolates from wheat rhizosphere were able to produce HCN. The results indicate that most of the bacteria, tested in the experiment, have plant growth-promoting activities. This is the first time that such PGPR species are isolated from the Iranian soils. With respect to their great biological capacities they can be used for wheat and canola inoculation in different parts of the world, which is of very important agricultural implications.     

Isolation and identification of endophytic bacteria from root tissues of Salvia miltiorrhiza Bge. and determination of their bioactivities

Endophytic bacteria are microorganisms that live in host plants, but do not cause diseases to the hosts. This study examined the occurrence, distribution, growth-promoting and antifungal activities of endophytes in the root of Salvia miltiorrhiza Bge. Six endophytic bacterial strains, which belong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolated from the root of healthy S. miltiorrhiza. Cell suspension (approx. 10⁹ cell?·?ml^?1) of two isolates and cell-free fermentation filtrate of four isolates substantially promoted the growth of hypocotyl and radicle of muskmelon seeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on tested pathogenic fungi, namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. Six compounds were isolated from one of the six endophytic bacteria, namely, Bacillus aryabhattai, and two of these compounds displayed certain antifungal activity against three tested S. miltiorrhiza pathogens. Our work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and that associated bacterial isolates have growth-promoting effect on muskmelon seeds and are expected to be a potential source for bioactive metabolites.