Isolation of human colonic fibrolytic bacteria

Bacteria able to degrade pebble-milled filter paper cellulose (PMC) were detected in only two of five human faecal samples and at very low concentrations ( ca 100/g faeces). For another sample, the most probable number of cellulolytic organisms was 8·7 times 10⁸/g with pebble-milled cell walls of New Zealand spinach as substrate, whereas PMC was not degraded. The cellulolytic bacteria isolated from those samples have been tentatively identified as atypical Ruminococcus spp. (two strains) and Eubacterium sp. A hemicellulolytic Butyrivibrio fibrisolvens was also isolated.     

Isolation and characterization of halophilic Archaea able to produce biosurfactants

Halotolerant microorganisms able to live in saline environments offer a multitude of actual or potential applications in various fields of biotechnology. This is why some strains of Halobacteria from an Algerian culture collection were screened for biosurfactant production in a standard medium using the qualitative drop-collapse test and emulsification activity assay. Five of the Halobacteria strains reduced the growth medium surface tension below 40 mN m⁻¹, and two of them exhibited high emulsion-stabilizing capacity. Diesel oil-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 35% sodium chloride or up to 25% ethanol in the aqueous phase. Emulsions were stable to three cycles of freezing and thawing. The components of the biosurfactant were determined; it contained sugar, protein and lipid. The two Halobacteria strains with enhanced biosurfactant producers, designated strain A21 and strain D21, were selected to identify by phenotypic, biochemical characteristics and by partial 16S rRNA gene sequencing. The strains have Mg²⁺, and salt growth requirements are always above 15% (w/v) salts with an optimal concentration of 15–25%. Analyses of partial 16S rRNA gene sequences of the two strains suggested that they were halophiles belonging to genera of the family Halobacteriaceae, Halovivax (strain A21) and Haloarcula (strain D21). To our knowledge, this is the first report of biosurfactant production at such a high salt concentration.     

Isolation of soil bacteria able to hydrolyze both organophosphate and carbamate pesticides

Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae able to rapidly degrade the organophosphate (OP) fenamiphos (FEN) were isolated. Denaturating gradient gel electrophoresis analysis revealed that the two isolates were dominant members of the enrichment culture. Clone libraries further showed that bacteria belonging to α-, β-, γ-proteobacteria and Bacteroidetes were also present in the final enrichment but were not isolated. Both strains hydrolyzed FEN to fenamiphos phenol which was further transformed, only by P. putida. The two strains were using FEN as C and N source. Cross-feeding studies with other pesticides showed that P. putida degraded OPs with a P-O-C linkage and unexpectedly degraded the carbamates oxamyl and carbofuran being the first wild-type bacterial strain able to degrade both OPs and carbamates. The same isolate exhibited high bioremediation potential against spillage-level concentrations of aged residues of FEN and its oxidized derivatives.     

Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.     

Isolation and characterization of a fungus able to degrade pyrethroids and 3-phenoxybenzaldehyde

Fungal strain HU, isolated from activated sludge and identified as a member of the genus Cladosporium based on morphology and sequencing of 28S rRNA, was shown to degrade 90% of fenvalerate, fenpropathrin, β-cypermethrin, deltamethrin, bifenthrin, and permethrin (100 mg L⁻¹) within 5 days. Fenvalerate was utilized as sole carbon and energy source and co-metabolized in the presence of sucrose. Degradation of fenvalerate occurred at pH 5-10 at 18-38 °C. The fungus first hydrolyzed the carboxylester linkage to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde, and subsequently degraded these two compounds with a q_max, K_s and K_i of 1.73 d⁻¹, 99.20 mg L⁻¹ and 449.75 mg L⁻¹, respectively. Degradation followed first-order kinetics. These results show that the fungal strain may possess potential to be used in bioremediation of pyrethroid-contaminated environments.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

猪源乳酸菌的分离、鉴定及其生物学特性

【目的】将分离自猪肠道粘膜、食糜和粪便的乳酸菌,通过产乳酸能力、生长性能、耐酸和耐胆盐性能及抑菌能力评价,筛选适应养猪生产的潜在益生特性的菌株。【方法】共分离获得155株乳酸菌纯菌株,从中筛选出4株产酸能力较强的乳酸菌,结合生理生化试验及细菌16S rRNA测序鉴定其种属,评价候选乳酸菌的生长情况、耐酸、耐胆盐及抑菌特性。【结果】综合变色时间(8 h)、pH值(3.9)和乳酸含量(100 mmol/L),筛选出4株(L45、L47、L63和L79)候选菌株,经鉴定依次为罗伊氏乳杆菌、植物乳杆菌、约氏乳杆菌和粪肠球菌。该4株乳酸菌均可在体外快速生长;L47和L79能够耐受pH 2.5的酸性环境,L47能够耐受0.5%胆盐环境;各乳酸菌上清液与指示菌共培养,发现对E coli K88和沙门氏菌均产生了抑制作用,其中L47上清液对指示菌的抑制作用较强。【结论】L47具有较好的产酸性能与生长性能、可耐受猪胃酸和肠道胆盐环境,对E.coli K88和沙门氏菌具有较好的抑制作用,说明该乳酸菌具有潜在的益生特性。     

Characterization of mutants of Sulfolobus solfataricus signature amidase able to hydrolyse R-ketoprofen amide

The amidase from Sulfolobus solfataricus enantioselectively hydrolyzes S-ketoprofen amide to its corresponding acid. We identify three independent SsAH mutants that hydrolyze R-ketoprofen amide and built computational models of their three-dimensional structure. Interestingly the mutations do not specifically affect residues near the active site, or directly interacting with the substrate.     

Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.     

Lactic acid bacteria isolated from apples are able to catabolise arginine

We investigated the potentiality of lactic acid bacteria (LAB) isolated from two apples variety to utilize arginine at different initial pH values. Apples surface contained average levels of bacteria ranging from log 2.49 ± 0.53 to log 3.73 ± 0.48 cfu/ml for Red Delicious and Golden Delicious varieties, respectively. Thirty-one strains able to develop in presence of arginine at low pH were phenotypically and genotipically identified as belonging to Lactobacillus, Pediococcus and Leuconostoc genera. In general, they did not produce ammonia from arginine when cultivated in basal medium with arginine (BMA) at pH 4.5 or 5.2. When this metabolite was quantified only six strains belonging to Leuconostoc dextranicum, Lactobacillus brevis and Lactobacillus plantarum species formed higher ammonia amounts in BMA as compared to control. This was correlated with arginine utilization and it was more pronounced at pH 4.5 than 5.2. Analysis of citrulline production confirmed the arginine utilization in these bacteria by the arginine deiminase (ADI) pathway. Maxima citrulline production was observed for Lactobacillus brevis M15 at the two pH values. In this strain ammonia was formed at higher rate than citrulline, which was detected in concentration lower than 1 mM. Thus, main LAB species found on apple surfaces with abilities to degrade arginine by the ADI pathway under different conditions were reported here at the first time. The results suggested that the ADI pathway in apples LAB might not be mainly relevant for their survival in the acid natural environmental, despite leading to the ammonia formation, which may contribute to the increase in pH, coping the acid stress.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Isolation and characterization of oil-desulphurizing bacteria

The objective of this study was to isolate local bacterial strains capable of removing sulphur from oil fractions without degrading the hydrocarbon. Oil biodesulphurization is an important step in combating pollution problems emanating from burning fossil fuels. Organisms which survive on oil are plentiful in local Kuwaiti soils; however, those that selectively only attack the carbon–sulphur bond are more difficult to find. Three strains were isolated based on their ability to use dibenzothiophene (DBT) as a sole source of sulphur for growth at 30 °C. Similar to other biodesulphurization organisms, the strains convert DBT to [2-hydroxybiphenyl (2-HBP) as detected by gas chromatography (GC). The specific desulphurization activity was in the range 5–13 mol 2-HBP/g-cell × h. Identification of the strains, based on 16 rRNA gene sequence similarity, showed the strains to be Rhodococcus erythropolis and Rhodococcus globerulus. The biodesulphurization activity was enhanced by promoting oxidore-ductase enzyme co-expression through the addition of a carbon source. The desulphurization was limited by the availability of DBT to the organism. Interfacial mass transfer through the aqueous-organic layer was confirmed to be a limiting factor.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalysing the hydrolysis of chlorogenic acid

Among 15 Aspergillus strains, Aspergillus niger BRFM 131 was selected for its high chlorogenic acid hydrolase activity. The enzyme was purified and characterized with respect to its physico-chemical and kinetic properties. Four chromatographic steps were necessary to purify the protein to homogeneity with a recovery of 2%. Km of the chlorogenic acid hydrolase was estimated to be 10 microM against chlorogenic acid as substrate. Under native conditions, the protein presented a molecular mass of 170 kDa, and SDS-PAGE analysis suggested the presence of two identical 80 kDa subunits. Isoelectric point was 6.0; pH optimum for activity was determined to be 6.0 and temperature optima to be 55 degrees C. The N-terminal sequence did not present any homology with other cinnamoyl ester hydrolases previously described suggesting the purification of a new protein. The chlorogenic acid hydrolase was used successfully for the production of caffeic acid, which possesses strong antioxidant properties, from natural substrates specially rich in chlorogenic acid like apple marc and coffee pulp.     

Peptidase activity of human colonic bacteria

Aminopeptidase activities of mixed faecal suspensions from four human donors and 12 of the most numerous species of human colonic bacteria were measured using alanine oligopeptides and various dipeptidyl- and amino acyl-arylamidase substrates. The pattern of hydrolysis of Ala(4)and Ala(5)in faecal suspensions, whereby Ala(2)was the first breakdown product, suggested that the main mechanism of peptide hydrolysis was dipeptidyl peptidase. Dipeptidyl p-nitroanilides and 4-methoxynaphthylamides were broken down more rapidly than amino acyl derivatives in three out of four individuals tested, consistent with this conclusion. The predominant Bacteroides spp. of the intestine also had greater dipeptidyl peptidase activity than amino acyl aminopeptidase activity, while Bifidobacterium, Clostridium, Enterococcus and Propionibacterium spp. had a more variable pattern of peptidase activities. Thus, peptide hydrolysis in the human intestine, as in the rumen, appears to be mainly a two-stage process which is initiated by dipeptidyl peptidases present in the most numerous Bacteriodes spp.     

产绿原酸内生菌的分离及其绿原酸合成途径关键基因的克隆和功能研究

【目的】分离产绿原酸的内生菌并对其绿原酸合成途径的一种关键酶基因进行克隆和功能研究。【方法】采用表面消毒法从金银花中分离内生菌,以高效液相色谱(HPLC)和液相色谱-质谱联用(LC-MS)筛选确定产绿原酸的内生菌,克隆、表达该内生菌的组氨酸解氨基酶(HAL)并进行酶活测定。【结果】从金银花根中分离到一株产绿原酸的内生细菌RP1,同时在该内生菌发酵液中检测到了绿原酸代谢的中间物肉桂酸。对RP1的分子鉴定表明该内生菌为枯草芽孢杆菌(Bacillus subtilis)。对RP1的HAL基因进行克隆和原核表达,酶活测定表明该酶具有HAL和PAL (苯丙氨酸解氨基酶)双功能,其PAL活性产生的肉桂酸与LC-MS检测的结果一致。【结论】推测该内生菌可能利用其HAL的苯丙氨酸解氨基活性将苯丙氨酸的催化产物肉桂酸导入苯丙烷途径,从而产生绿原酸。