净化养殖水体紫色非硫光合细菌的筛选与鉴定

本研究从杭州养鱼塘水样及底泥样品中富集分离得到三株紫色非硫光合细菌,分别命名为HZ-3,HZ-4,HZ-5。 通过比较这三株菌对鱼、虾养殖水体的净化效果,筛选出菌株HZ-5 净化能力较强 ,经过5天的处理,该菌株使养鱼塘水样的COD 降低24.87%;使养虾池塘水样的COD 降低 36.99%;使养鱼塘水样的亚硝态氮的降解率达到97.79%。对菌株HZ-5进行了形态学观察、生理生化鉴定、活细胞吸收光谱以及16S rDNA序列分析。16S rDNA序列分析结果表明该菌株与沼泽红假单胞菌的16S rDNA序列有高达99％的同源性,结合形态特征和生理生化特性以及活细胞吸收光谱特征等,将其鉴定为沼泽红假单胞菌（Rhodopseudomonas palustris）。     

Sequence analysis of partial toxR gene from Philippine Vibrio isolates and design of toxR-targeted primers for detection

Vibriosis in penaeid species cultured in the Philippines results in massive mortalities and consequently in severe economic losses in the shrimp industry. Rapid and accurate detection of the causative agent of the disease is imperative. In this study, toxR gene sequence analysis of ten Vibrio isolates (from several provinces of the Philippines) implicated in disease affecting the penaeid shrimp (Penaeus monodon) was performed in order to develop a toxR-targeted PCR detection of similar strains of shrimp pathogens. Analysis of the partial toxR gene revealed 97-100% sequence similarity among the ten Philippine Vibrio isolates. Distinct sequence variation of the toxR gene, however, was observed between the Philippine Vibrio isolates and the type strains, with the Philippine isolates exhibiting only 92-93% and 74-75% sequence similarity with the type strain V. campbellii (NBRC 15631T) and V. harveyi (NBRC 15634T), respectively. The use of a PCR primer set that was designed based on toxR sequences of the Philippine Vibrio isolates amplified the expected 226-bp toxR fragment using templates from all ten Philippine Vibrio isolates. No amplified product was observed in PCR using templates from type strains of V. harveyi, V. campbellii, and other non-target bacteria, suggesting that the primers were specific for the Philippine Vibrio isolates. The toxR-targeted PCR primers reported in this study could be useful in the detection of Philippine Vibrio isolates associated with mortalities in the shrimp industry, which could not be detected in PCR using primers designed for type strains of V. harveyi and V. campbellii.     

Microbial Dissimilatory Sulfur Cycle in Acid Mine Water

Ferric, sulfate, and hydrogen ions are produced from pyritic minerals associated with coal as a result of autotrophic bacterial metabolism. Water carrying these ions accumulated behind a porous dam composed of wood dust originating at a log-cutting mill. As water seeped through the porous dam, it was enriched in organic nutrients which then supported growth and metabolism of heterotrophic bacteria in the water downstream from the dam. The heterotrophic microflora within and below the sawdust dam included dissimilatory sulfate-reducing anaerobic bacteria which reduce sulfate to sulfide. The sulfide produced caused the chemical reduction of ferric to ferrous ion, and black FeS precipitate was deposited on the pond bottom. A net increase in the pH of the lower pond water was observed when compared to the upper pond water. Microbial activity in the wood dust was demonstrated, and a sequence of cellulose degradation processes was inferred on the basis of sugar accumulation in mixed cultures in the laboratory, ultimately yielding fermentation products which serve as nutrients for sulfate-reducing bacteria. Some of the microorganisms were isolated and characterized. The biochemical and growth characteristics of pure culture isolates were generally consistent with observed reactions in the acidic environment, with the exception of sulfate-reducing bacteria. Mixed cultures which contained sulfate-reducing bacteria reduced sulfate at pH 3.0 in the laboratory with sawdust as the only nutrient. Pure cultures of sulfate-reducing bacteria isolated from the mixed cultures did not reduce sulfate below pH 5.5.     

Numerical taxonomy and ecology of petroleum-degrading bacteria.

A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 of the petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

一株紫色非硫光合细菌净化养殖水体初步研究

选取从杭州养鱼塘水样中分离得到的1株紫色非硫光合细菌菌株HZ-1,利用它的纯培养物处理养殖水体,测定COD去除率和亚硝态氮降解率。结果表明,菌株HZ-1可以有效地分解水体中的污染物,去除水体中的COD,对养鱼塘水去除率达到20.99%;对养虾池水去除率达到了36.09%;菌株HZ-1可以有效地降低水体的亚硝态氮,对养鱼塘水亚硝态氮的降解率为41.18%,具有较好净化效果。     

Efficient decomposition of shrimp shell waste using <Emphasis Type="Italic">Bacillus cereus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis>

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37°C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37°C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.     

Numerical Taxonomy Analysis of Bacteria Isolated from the Completed 'Most Probable Numbers' Test for Coliform Bacilli

Lactose-fermenting bacteria were isolated from oyster, water and sediment samples tested to determine the Most Probable Number (MPN) of coliforms present. The completed test series was used to enumerate the total number of coliforms. The strains from the completed tests of the MPN analysis were compared with named reference cultures, for which 100 phenotypic characters were recorded and the taxonomic data analysed by methods of numerical taxonomy. The isolates were recovered in 13 phenetic groups, defined at or above the 80% similarity level. Nine phena were identified as Acinetobacter calcoaceticus, Chromobacterium violaceum, Citrobacter freundii, Erwinia herbicola, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, Serratia liquefaciens , and Ser. marcescens. Of these, Ac. calcoaceticus and C. violaceum were isolated exclusively from oyster tissue samples and strains of the other taxa from both water and oyster samples. Some strains of Esch. coli, Er. herbicola, H. alvei and bacteria that were not Enterobacteriaceae were found to lose the ability to ferment lactose, following storage on laboratory media. Identifications from diagnostic keys, rapid identification systems and IMViC patterns were not always in agreement.     

<Emphasis Type="Italic">Bacillus cereus</Emphasis> is a microbial decomposer of 2,4-dichlorophenol

A microorganism degrading 2,4-dichlorophenol isolated from an aeration pond of the Baikal Pulp and Paper Mill was identified as Bacillus cereus BIP507 based on morphological and physiological characters as well as 16S rDNA sequencing. This microorganism proved able to degrade high 2,4-dichlorophenol concentrations (up to 560 μM).     

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.     

Physiological characterization of heterotrophic bacterial communities from selected aquatic environments

Three different biotopes, groundwater, surface water, and activated sludge, were examined and the total colony count on nutrient agar determined. The bacteria that could be isolated from the agar plates were identified and their in vitro activities investigated. Three principal approaches were used: (1) isolates were identified and the results used in a numerical analysis to determine their similarity; (2) the different physiological properties of isolates originating from a single biotope were compiled and used to characterize the community (collective total activity); and (3) the diversity of the physiological properties of the isolates of all populations was determined; on the basis of main characters a heterotrophic diversity index was calculated. The possibility of using a polyphasic ecological study to characterize aquatic bacterial communities is considered.     

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys,Antarctica

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.     

The effects of pond duration on the life history traits of an ephemeral pond crustacean,Eulimnadia texana

We examined the relationship between pond duration and life history characters of the clam shrimp Eulimnadia texana, a species inhabiting ephemeral ponds in southwestern North America. Since the shrimp live in temporary habitats, we predicted that there should be high selection pressure on life history characteristics associated with rapid development (e.g., fast growth, early maturity, etc.), rather than selection for increased longevity. Pond duration was estimated using a combination of average monthly rainfall and pond size (surface to volume ratio). Shrimp that live in smaller ponds (high surface to volume ratio)in areas with low average rainfall should, on average, experience a shorter total time available for development than those in larger ponds or in areas of higher rainfall. These shrimp should have an earlier age at maturity, reduced longevity, lower fecundity, and faster growth. Five replicate populations of clam shrimp were collected as cysts from five ponds. These shrimp were raised in a common garden experiment in the laboratory. Daily measurements of growth and egg production were taken and ages at maturity and death were recorded. Shrimp from areas with higher average rainfall had slower growth, higher fecundity, greater longevity, and an earlier age at maturity than those from areas with lower average rainfall. If average rainfall is an accurate measure of pond duration, then the first three of these life history traits differ in the directions expected. However, age at maturity varied in a manner opposite to that expected, being earlier in the ponds with longer duration. Surface to volume ratio was not helpful in further resolving differences in these life history characters. This revised version was published online in August 2006 with corrections to the Cover Date.     

掘氏疫霉遗传学研究 Ⅰ.自交后代(S_1)生物学性状的遗传与变异

测定了掘氏疫霉Phytophthora drechsleri Tucker生长速率、菌落形态、菌体形态、产孢能力、耐35℃高温生长、致病力等生物学性状在自交S_1代的遗传与变异。2个掘氏疫霉野生型菌株(A_1、A_2各1株)经聚碳膜间隔配对诱导自交产生的卵孢子(保存110天左右)在S+L培养基上萌发,分别获得30和35个单卵孢株(萌发率10%左右)。测定结果表明,上述生物学性状在2个亲本自交后代均发生变异,部分单卵孢株生长速度减慢、菌落形态明显改变、孢子囊和菌丝畸形、产孢能力增强、在35℃中弱生长或不生长,或致病力减弱直至无致病力。在亲本的单游动孢子无性系后代未观察到上述变异,说明上述生物学性状变异主要因亲本遗传因子的杂合性所致。从遗传学角度探讨了在自然条件下掘氏疫霉生物学性状的变异性以及某些分类依据不稳定的原因,指出有性生殖过程中的基因重组可能是引起掘氏疫霉生物学性状变异的主要原因之一。     

Genetic polymorphism by RAPD-PCR and phenotypic characteristics of isolated thermotolerant Bacillus strains from hot spring sources

The polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) assay, morphological, physiological, biochemical and antimicrobial susceptibility test methods have been evaluated for use in the taxonomy of isolated thermotolerant Bacillus from Jordanian hot springs, with specific reference to strains Geobacillus stearothermophilus (ATCC 12980), Bacillus circulans (ATCC 4513) and Bacillus sphaericus (ATCC 14577). A RAPD assay has been optimized and is able to discriminate between numerous thermotolerant Bacillus strains. RAPD-PCR was found to give reproducible thermotolerant Bacillus strains classification of DNA fingerprints for 14 strains including 3 reference strains. A study of 14 isolates and 3 reference strains, analyzing 53 phenotypic characters, resulted in their allocation to five major clusters at 60% similarity. Whereas at 80% similarity, twelve taxonomically distinct groups were evident.     

Vibrio alginolyticus associated with white spot disease of Penaeus monodon

In February 2000, white spot disease outbreaks occurred among cultured Penaeus monodon in extensive shrimp farms on the southwest coast of India. Bacteria were isolated from infected shrimp that showed reddish body coloration and white spots in the cuticle. The isolates were screened on thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. The primary isolate (QS7) was characterized as V. alginolyticus based on morphological, biochemical and physiological characteristics. Antibiotic sensitivity tests of QS7 indicated that the isolate was highly sensitive to chloramphenicol, ciprofloxacin, nalidixic acid and streptomycin. Pathogenicity tests confirmed that the isolate was virulent for P. monodon. Based on the lethal dose (LD50) value (5 x 10(6) cfu per shrimp), it was inferred that shrimp weakened by white spot syndrome virus would succumb to secondary infection by QS7.     

Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface

Ten bacterial strains were isolated from alkylpyridine polluted sediments 7.6 m below the surface. These strains were able to degrade 11 different alkylpyridine isomers. Degradation rates depended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were more resistant to microbial attack. Of the 10 strains, 6 isolates were selected for detailed study. These isolates mineralized the isomers to CO2, NH4+, and biomass. All strains were gram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemical properties revealed similarity between strains. Eeach strain however, had a limited substrate range which enabled it to degrade no more than 2 to 3 compounds of the 14 alkylpyridine isomers tested. Examination of the genetic variability among cultures with the randomly amplified polymorphic DNA technique revealed high levels of genomic DNA polymorphism. The highest similarity between 2 strains (0.653) was observed between 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substrate specificity is under investigation.     

Numerical taxonomy of heavy metal-tolerant bacteria isolated from an estuary.

A total of 230 strains of metal-tolerant bacteria from water and sediment samples collected in Chesapeake Bay were isolated on a medium containing cobalt, lead, mercury, or molybdenum. In addition, a set of 71 cultures were simultaneously isolated on glucose tryptone yeast extract agar medium without metals. Twenty-three reference strains were also included in the numerical taxonomy study of these bacteria, bringing the grand total of strains examined to 324. All strains were examined for 112 biochemical, cultural, morphological, and physiological characters. The taxonomic data obtained were analyzed by computer and the simple matching (SSM) and Jaccard (SJ) coefficients were calculated. Clustering achieved by unweighted average linkage is presented and, from sorted similarity matrices and dendrograms, 294 strains, i.e., 97% of the total, were recovered in 12 phenetic groups defined at the 75 to 80% similarity level. Among the strains there were nine phena presumptively identified as Bacillus, Erwinia, Mycobacterium, Pseudomonas, and coryneforms. From the results of the taxonomic study, it is concluded that metal tolerance in estuarine water and sediment bacteria occurs among a restricted range of taxa distributed throughout the estuarine environment.     

Ribotyping of Pseudomonas aeruginosa isolates from patients and water springs and genome fingerprinting of variants concerning mucoidy

Abstract Thirty-one strains of Pseudomonas aeruginosa , isolated from water springs, clinical isolates (some of which were from cystic fibrosis (CF) patients), and two type cultures, were characterized by ribotyping. After restriction of chromosomal DNA of the different isolates with Eco RI and hybridization of Southern transfer blots with 2-acetylaminofluorene labelled Escherichia coli 16S + 23S rRNA probe, eleven different ribopatterns were obtained, representing variations of a dominant profile. This largely predominant pattern included both type cultures, all six isolates from water springs, 33% of the nine CF isolates and 43% of fourteen other clinical isolates most of them from nosocomial infections. When the genomic macrorestriction fingerprints of three mucoid CF isolates, with Ase I, Dra I or Bfr I were compared with those of their spontaneous variants, concerning mucoidy, no differences were detected.