Isolations and Identifications of Two Psychrotrophs and Preliminary Research on Their Applications

[Objective] The research aimed to initially study degradation effect of the CODc, in sewage by two psychrotrophs. [Method] Two psychrotrophs were isolated from the activated sludge of wastewater treatment plant in Tianjin Konggang Economic Area. CODc, degradation ability of the screened psychrotroph was analyzed in simulated domestic wastewater at 6℃. [Result] K 36 was identified as Comamonas testosterone, and K 38 was identified as Serratia fonticola. CODcr degradation abilities of the two strains were different in test. COOcr removal rates of the K 36 and K 38 respectively reached up to 23% and 53%. The measured result of growth rate suggested that two psychrotrophs both had high activities at low temperature. [ Conclusion] K 36 and K 38 had potentials in wastewater treatment application.

应用耐冷菌株改善寒冷地区冬季人工湿地系统生物脱氮效果

通过探索性实验,确定了三种耐冷菌培养基,分别用于培养耐冷细菌、耐冷放线菌和耐冷霉菌.培养出的耐冷菌株在6℃条件下被分离纯化,然后通过一系列实验分别鉴定它们的生理特性并检验它们的脱氮能力,并测定pH值、温度对脱氮效果的影响规律.实验结果显示pH值为7～8时,各菌株生长得最好;生物荧光层析光谱实验发现三种菌株都在15℃左右活性最大,0℃以下仍有一定的活性,高于35℃时,基本失去正常的代谢能力.当实验菌液投加量为5.0%,实验温度为6℃,三种耐冷菌对氨氮的去除率分别为57.7%、59.0%及58.7%,相同条件下,投加混合菌种可使氨氮的去除率提高到67.2%.

北方人工湿地耐冷菌株对模拟污水的处理效果

[目的]研究北方人工湿地耐冷菌株对模拟污水的处理效果。[方法]在冬季低温条件下采集人工湿地污泥,从中分离出6株耐冷菌株(编号为菌株A、B、C、D、E和F),并筛选出综合去除效果最好的菌株,确定该菌株的最佳生长条件。[结果]当温度为8℃时,6株耐冷菌对COD、总磷和氨氮的去除率分别为11.27%～62.92%、7.16%～61.20%和3.86%～58.63%,其中菌株E对该废水的净化效果最好。菌种E的最适生长温度为16℃,在最适生长温度下菌株E对COD、总磷和氨氮的去除率有所升高,分别为8℃时的1.20、1.25、1.02倍。[结论]菌株E在湿地污水处理冬季运行中具有良好的应用前景。

Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea,Arctic

Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the bio-technological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5) , producing cellulose at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 351 or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes including cellulase, amylase, agar hydrolase and protease, at low or moderate temperature conditions, which is certainly of it potential value for applications.

Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria

A study was conducted to compare the diversity of 2-, 3-, and 4-chlorobenzoate degraders in two pristine soils and one contaminated sewage sludge. These samples contained strikingly different populations of mono-chlorobenzoate degraders. Although fewer cultures were isolated in the uncontaminated soils than contaminated one, the ability of microbial populations to mineralize chlorobenzoate was widespread. The 3- and 4-chlorobenzoate degraders were more diverse than the 2-chlorobenzoate degraders. One of the strains isolated from the sewage sludge was obtained. Based on its phenotype, chemotaxonomic properties and 16S rRNA gene, the organism S-7 was classified as Rhodococcus erythropolis. The strain can grow at temperature from 4 to 37℃. It can utilize several （halo）aromatic compounds. Moreover, strain S-7 can grow and use 3-chlorobenzoate as sole carbon source in a temperatures range of 10-30℃ with stoichiometric release of chloride ions. The psychrotolerant ability was significant for bioremediation in low temperature regions. Catechol and chlorocatechol 1,2-dioxygenase activities were present in cell free extracts of the strain, but no （chloro）catechol 2,3- dioxygenase activities was detected. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-chlorobenzoate. On the basis of these results, we proposed that S-7 degraded 3-chlorobenzoate through the modified ortho-cleave pathway.

An Approach to the Identification and Characterisation of a Psychrotrophic Lipase Producing <i>Pseudomonas</i>sp ADT3 from Arctic Region

The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A novel psychrotrophic bacterial strain showed good growth on minimal medium containing lipid as the only carbon source. Microbiological characterisation of the isolate showed that it was a gram negative rod. The strain was tested for the production of extracellular lipase enzyme. The enzymes were partially purified by 90% saturated ammonium sulfate and dialysis for desalting. The bacterium was identified as Pseudomonas sp ADT3 by 16S rRNA amplification and sequencing which had been deposited in the NCBI GenBank with accession number JX914667. Phylogenetic tree was also constructed with MEGA5 software and showed the highest level of sequence similarity with Pseudomonas sp. HC3-13 strain. The microorganism had a growth optimum at pH 8.0 and temperature 22°C. Optimization of different parameters e.g. temperature, pH, incubation time, cofactors etc. was performed for the extracellular lipase activity. The hydrolytic activity of the enzyme was enhanced 5 times by Pb2+ but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. For the molecular weight estimation of enzyme SDS-PAGE was done which showed an inducible band of approximately 13.9 KDa. Activity staining and mass spectrometry techniques were also performed.

Relation between Mesophilic and Psychrotrophic Aerobic Sporulating Microorganisms in Milk

The occurrence and the detection system of mesophilic and psychrotrophic aerobic sporulating microorganisms （MPAS） in raw cow＇s milk were studied. Samples of raw cow＇s bulk tank milk were taken 21 times in 14 farms during one year. Basis of the method for MPAS assessment is the milk sample inactivation at the temperature 80-82 ℃ for 30 minutes followed by incubation in cultivation dishes at 30 -4- 1 ℃ for 3 days-mesophilic aerobic sporulates （MAS）, and at 6.5 .4- 1 ℃ for 10 days-psychrotrophic aerobic sporulates （PAS）. The total count of MPAS was within the span 2.5-340 CFU/mL （colony forming units）. Average plate count of MPAS was 59.4 CFU/mL with variation coefficient 93.1%. MPAS count found in the same dishes at incubation for mesophilic and subsequently strictly psychrophilic microorganisms （MAS ＋ SPAS） which enables to exclude overestimation of results by sporulates able to grow at both incubation temperatures was on average 56.9 CFU/mL what represents 95.8% out of the number of sums of individual dishes at two temperatures （MAS ＋ PAS）. Correlation coefficient of these two types of results r = 0.99 gives evidence of close dependence that is expressed by linear regression equation y = 0.9773x. We can consider the method using at first 30 .4- 1 ℃ and subsequently 6.5 -4-1℃ （MAS ＋ SPAS） as more correct than the method with the opposite order of cultivation temperatures because of better regression coefficient of linear dependence and higher correlation coefficient in relation to the sum of independent incubations （MAS ＋ PAS）.

Diversity and cold adaptation of endophytic fungi from five dominant plant species collected from the Baima Snow Mountain,Southwest China

The diversity and cold adaptation of endophytic fungi associated with five dominant plant species collected from the Baima Snow Mountain(altitude 4,000–4,300 m),Southwest China,were investigated.A total of 604 culturable endophytic fungi were isolated from 800 stems and leaf segments.The colonization rate of endophytic fungi in the five plant species ranged from 54%to 91%,and the colonization rate of endophytic fungi in the stems was significantly higher(87%)than that in the leaves(67%)(X∼2045.172,P<0.001,chi-squared test).Based on the morphological characteristics and the ITS sequence analysis,the isolates were identified to 43 taxa,of which Cephalosporium,Sirococcus,Penicillium and Aspergillus were the dominant genera,and their relative frequencies were 14.06%,12.58%,9.44%and 7.95%,respectively.The Shannon-Weiner diversity indices and the Sorenson’s coefficient similarity indices of the endophytic fungi from the five plant species ranged from 1.25 to 2.70 and 0.53 to 0.67,respectively.Growth temperature tests indicated that 75%of the isolates from the Baima Snow Mountain were psychrotrophs and 14%were the transitional type between psychrotrophs and mesophiles,whereas all of the isolates from the Yuanjiang Dry-hot Valley were mesophiles,which suggested that the endophytes from the Baima Snow Mountain possess a remarkable ability to adapt to cold environments.

Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabUities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature （28 ℃） and refrigerated temperature （7 ℃）. Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens （d）. Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive ^-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 ℃, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.