膜微阵列技术的建立及检测细菌条件的优化

目的 探讨膜微阵列技术检测细菌的可行性。方法 在细菌的 1 6SrRNA基因内设计了通用引物和特异性探针 ,将探针点在膜上制备微阵列 ,再与地高辛标记的DNA杂交 ,比较了随机引物标记法和PCR掺入标记法的灵敏度 ,探讨了硝酸纤维素膜和尼龙膜作基片的可行性 ,并比较了不同的固定条件和杂交条件下的杂交结果 ,以标准菌株DNA为样本 ,建立起微阵列技术。结果 随机引物标记法和PCR掺入标记法的灵敏度均为 0 .1pg ;尼龙膜较之硝酸纤维素膜可更好的结合寡核苷酸 ,经紫外线交联 3min ,与杂交液 3(5×SSC ,5g/LSDS ,1 %封闭剂 )在 5 5℃杂交结果最好。标准菌株除结核杆菌、变形杆菌外 ,均能与其对应的特异探针杂交。结论 利用膜微阵列技术检测细菌有快速、敏感、特异。

水稻上两类人体条件致病细菌的鉴别与检测

洋葱伯克氏菌(Burkholderiacepacia,Bc)和铜绿假单胞菌(Pseudomonas aeruginosa,Pa)既是农业上的生防菌又是医院的人体条件致病菌,了解它们的特征及在水稻上的分布情况是有效管理和利用这类原核生物的关键。在过去的9年中,利用常规细菌学、致病性测定、Biolog和脂肪酸分析(FAME)对采自平原、丘陵和山区稻区的631份稻谷样本和117份稻株样本进行了两类人体条件致病细菌的鉴定和检测研究。Bc菌和Pa菌可通过培养性状和常规细菌学方法作初步鉴别,Biolog和FAME能把它们鉴定到种,但Bc菌的基因型难以区分。Bc菌和Pa菌主要存在于水稻的根部;Bc菌和Pa菌在浙江的平原稻区分布最广,分别达6.1%和16.1%;在山区稻区最低,分别为1.0%和7.8%。

非常条件下存活细菌的检测及其卫生学意义

按照常法检测存在于食品，空气，水体等外环境中的活菌，是评价其污染度和安全性的重要依据之一，然而，随着科技的发展和卫生质量要求的提高，一些在外环境中存活而不易为常规检测所发现的细菌，对人们的健康构成了条件性威胁，换言之，这些菌可在某种条件下生长，繁殖，致病，由于这类菌产生的背景不同，作者暂且概称为非常条件下存活的细菌，包括受伤细菌，饥饿存活细菌和非可培养细菌等，现将这类细菌的研究进展综述如下。

细菌生长条件和细菌吸附在氧化铁硫杆菌生物浸出黄铜矿中的作用

本文探讨了氧化铁硫杆菌的生长条件和细菌吸附在浸出黄铜矿中的作用．在比较试验中采用了生长于硫、硫代硫酸盐和亚铁离子基质的氧比钱硫杆菌．培养于硫这一固体基质的细菌，需要一种细菌吸附力，该力与无机盐介质中有可溶性硫代硫酸盐和亚铁离子存在时所需的那种力不同．培养于固体基质的细胞呈现出比那些培养于液体基质的细胞更高的浸出率．研究发现利用固体基质培养的细胞在浸出时，不会出现液体基质培养的细胞在浸出过程中初期的粘滞阶段．

Cultural and luminescent Conditions of a Marine luminous Bacterium

Study of marine noctilucence in marine is important to fishery, environmental monitoring and military affairs. A luminous bacterial strain D2 was isolated from the marine sediment samples collected near Donghai Island in Zhanjiang, China. The primary cultural and luminescent conditions of luminous bacterium D2 which was identified as Vibrio sp. were determined in liquid culture. The results showed that pH 7.0, 35 ℃, with 2.0 % NaCI, were the best growth conditions, and pH5 - 6, 20℃, OD600 0.08, with 3.0 % NaCI, were the optimal luminescent conditions.

Optimization of Fermentation Medium and Conditions of Antibiotic Active Substances Produced by Antarctic Psychrotrophic Bacterium Rheinheimera sp. 97

[Objective] The aim was to optimize the fermentation medium and conditions of antibiotic active substances produced by Antarctic psychrotrophic bacterium Rheinheimera sp.97.[Method] Single-factor experiment and orthogonal test were adopted to optimize the fermentation medium of antibiotic active substances produced by Antarctic psychrotrophic bacterium R.sp.97,while the fermentation conditions were optimized by single-factor experiment.[Result] The optimum fermentation medium for the antibiotic active substances production was as follows：tryptone 3.0 g/L,ammonium sulfate 1.0 g/L,starch 2.0 g/L,NaCl 15.0 g/L.The optimized fermentation conditions were as follows：the starting pH of medium was 8.0,fermentation temperature was 10 ℃,liquid volume in Erlenmeyer flask was 30 %（V/V）and inoculation amount was 1%（V/V）.Under the optimized fermentation medium and conditions,the antibacterial activity of R.sp.97 was increased by 18.1%.[Conclusion] This study had provided basis for the antibiotic active substances produced by Antarctic psychrotrophic bacterium R.sp.97.

Bioleaching of low-grade copper sulfide ores by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

The grown conditions of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were investigated,and then experiments were conducted to research the bioleaching behaviors of crude ore of copper sulfide and hand-picked concentrates of chalcopyrite by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans.The experimental results show that the bacteria grow best when the temperature is(30±1) °C and the pH value is 2.0.The bacteria concentration is 2.24×107 mL-1 in this condition.It is found that the copper extraction yield is affected by the inoculum size and the pulp density and the extraction yield increases as the inoculum size grows.The bioleaching rates reach their highest point in sulfide copper and chalcopyrite with a pulp density of 5% and 10%,respectively.Column flotation experiments of low-grade copper sulfide ores show that the bioleaching recovery reaches nearly 45% after 75 days.

Optimization of Culture Conditions and Medium Composition for the Marine Algicidal Bacterium Alteromonas sp. DH46 by Uniform Design

Harmful algal blooms(HABs) have led to extensive ecological and environmental issues and huge economic losses.Various HAB control techniques have been developed,and biological methods have been paid more attention.Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner,and kill or damage the algal cells.A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp.The culture conditions were optimized using a single-factor test method.Factors including carbon source,nitrogen source,temperature,initial pH value,rotational speed and salinity were studied.The results showed that the cultivation of the bacteria at 28℃ and 180 r min-1with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46.The optimal medium composition for strain DH46 was determined by means of uniform design experimentation,and the most important components influencing the cell density were tryptone,yeast extract,soluble starch,NaNO3 and MgSO4.When the following culture medium was used(tryptone 14.0g,yeast extract 1.63g,soluble starch 5.0 g,NaNO3 1.6 g,MgSO4 2.3 g in 1L),the largest bacterial dry weight(7.36 g L-1) was obtained,which was an enhancement of 107% compared to the initial medium;and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.

Statistical approach for the culture conditions optimization of magnetotactic bacteria for magnetic cells production

The culture of Magnetospirillum magneticum WM-1 depends on several control factors that have great effect on the magnetic cells concentration. Investigation into the optimal culture conditions needs a large number of experiments So it is desirable to minimize the number of experiments and maximize the information gained from them. The orthogonal design of experiments and mathematical statistical method are considered as effective methods to optimize the culture condition of magnetotactic bacteria WMol for high magnetic cells concentration. The effects of the four factors, such as pH value of medium, oxygen concentration of gas phase in the serum bottle, C：C （mtartaric acid： m=succinic acid） ratio and NaNO3 concentration, are simultaneously investigated by only sixteen experiments through the orthogonal design L16（44） method. The optimal culture condition is obtained. At the optimal culture condition （ pH 7.0, an oxygen concentration 4.0%, C：C （mtartaric acid： m=succinic acid） ratio 1：2 and NaNO3 100 mg 1^-1）, the magnetic cells concentration is promoted tO 6.5×10^7 cells ml^-1, approximately 8.3% higher than that under the initial conditions. The pH value of medium is a very important factor for magnetic cells concentration. It can be Proved that the orthogonal design of experiment is of 90% confidence. Ferric iron uptake follows MichaelisoMenten kinetics with a Km of 2.5 pM and a Vmax of 0.83 min^-1.

Isolation of a Halophilic Bacterium, Bacillus sp. Strain NY-6 for Organic Contaminants Removal in Saline Wastewater on Ship

The objective of this research was to examine if certain strains of Bacillus bacteria, could survive in dry powder products and if so, could the bacteria degrade organic contaminants in saline wastewater on a ship. As part of the study, we isolated 7 domesticated strains named NY1, NY2,..., and NY7, the strain NY6 showed to have the best performance for organic matter degradation and could survive in dry powder more than 3 months. NY6 was identified as Bacillus aerius, based on the morphological and physic-chemical properties. Its optimal growth conditions were as follows： salinity was 2%; temperature was 37℃; pH was in 6.5-7.0; best ratio of C： N： P was 100：5：1. The capability of its dry powder for Chemical Oxygen Demand （COD） removal was 800mg COD/g in synthesized marine wastewater with 2% salinity. The spores in the dry powder were 1.972×108 g-1.

Growth Response of Bacterial Antagonists in a Mix of Composted Wood Fibre Waste and Millet Seed under Sterile and Non-sterile Conditions

The potential use ofcomposted wood fibre waste （WFW） for the cultivation of bacterial antagonists of Sclerotinia minor was examined with the result that a mix of millet seed （20% w/w） and WFW, suitably amended with nutrients, proved to be an ideal matrix for the growth of some of these bacteria. Densities in terms ofcfu＇s ranged from 8.5 IOgl0 cfu/g dw to 10.5 logl0 cfu/g dw ullder sterile conditions after 14 days incubation. Lower population densities of the antagonists were achieved under non-sterile conditions in the compost： millet mix of between 7.9-9.3 logm cfu/g dw at the same period. However, when applied in a pot （glasshouse） trial to protect against S. minor, the millet seed appeared to stimulate the growth of this pathogen resulting in a high incidence of attack of lettuce plants after 2-3 weeks. Although the percentage of healthy seedlings increased following application of compost mix grown antagonists （at a rate of 5% v/v） when compared to the control treatment, these values were not statistically significant （p〉0.05） in most cases. Therefore, the use of millet seeds cannot be recommended as a nutrient supplement for the bacterial antagonist cultivation, if to be subsequently used to control fungal pathogens in the field.

Effect of chloride ion on bacterial pre-oxidation of arsenic-containing gold concentrate

The bacterial pre-oxidation process of arsenic-containing gold concentrates and the bacterial activity under different chloride ion concentrations were studied by using a mixture of thermophilic strains TCJ domesticated in production.The experimental result shows that with different samples and leaching systems,the adaptability and Cl- tolerance of bacteria are different,and that appropriate chloride ion concentration is conductive to bacterial oxidation,while higher chloride ion concentration will inhibit the bacterial activity and affect the pre-oxidation performance.Under the present production conditions,TCJ can adapt to the changes of water quality in the source of water and its critical chloride ion tolerance value is 2.7 g/L.

Methanethiol Removal from Biogas by Biological Conversion in an Anaerobic Biotrickling Filter

In this study, methanethiol（MT）-degradation bacteria were cultivated by using MT, methanol and trimethylamine as carbon sources under anaerobic conditions. It was found that the batch bacteria used MT and metha- nol as carbon sources grew faster than those used trimethylamine. The enriched bacteria used MT and methanol as the carbon sources were respectively inoculated in different biotrickling filters. The biological conversion performance ot MT under anaerobic conditions was investigated in biotrickling filters. The results showed that the performance of the biotrickling filter inoculated with the bacteria enriched using MT was better than that inoculated with the bacteria en- riched using methanol. When the inlet concentration of MT was 0.005vo1% （50 ppm）, the empty bed residence time was 50 s, pH value was 8.0, and the flow rate of the nutrient solution was 10 L/h, the removal efficiency of MT reached 95.3%. Adding methanol stimulated the growth of the biomass and the degradation of MT, but caused that some bacteria only degrading methanol outcompeted the bacteria only degrading MT. The concentration of sodium bicarbonate in the nutrient solution needed to be controlled lower than 30 g/L, otherwise, it would be harmful to the degradation of MT.

The Survival of Cefazolin Resistant Bacteria in Thermophilic Co-digestion of Dairy Manure and Waste Milk

Recent studies have suggested that there may be a link between the use of in-feed antibiotics and the prevalence of antibiotic-resistant bacteria in human infections. It is believed that anaerobic digestion is a potent method to reduce the antibiotic resistant bacteria present in waste from concentrated animal feeding operations. Cefazolin is a β-1actam antibiotic that is frequently used to treat the cows with mastitis in the Obihiro University herd. Disposal of untreated milk containing cefazolin residues promotes the occurrence of cefazolin resistant bacteria in the vicinity of farm, thus the objective of this study was to investigate the survival of antibiotic resistant bacteria in co-digestion of dairy manure and waste milk obtained from cows treated for mastitis with cefazolin under thermophilic conditions （55 ~C）. Cow manure, digested slurry and waste milk （cefazolin residue concentration 2.17 mg/L） were used as the materials in order to have three digester contents; 100% slurry, 50% slurry ＋ 50% manure and 50% slurry ＋ 45% manure ＋ 5% waste milk. The experiment was carried out using batch digesters （1 L） with active volume of 800 mL at 55 ℃ for 20 days to determine the survival of cefazolin resistant bacteria and to observe the digester performance by determining the bio gas and methane yield using gas chromatpgrapby. Dilution plate method was used to determine the population densities of total and cefazolin resistant bacteria at 0, 10th and 20th day of digestion. Total and cefazolin resistant bacterial counts were reduced with time by several orders until 10th day of digestion and those were almost similar at day 20th. Highest daily biogas and methane yield were observed in the digester contained slurry, manure and waste milk during early digestion period （until 5th day）. The results suggest that thermophilic co-digestion of dairy manure and waste milk would be a suitable technology for reducing antibiotic （cefazolin） resistant bacteria while obtaining better digester performance.

Establishment of Kinetics Models for Batch Fermentation Process of β-mannase with Bacillus licheniformis HDYM-04

In order to improve the yield of β-mannase and to investigate the rules of fermentation production, a high-yield β-mannase producing strain, Bacillus licheniformis HDYM-04, was used to investigate the kinetics models based on the optimal fermentation conditions： HDYM-04 strain was fermented at 37℃ for 30 h with agitation speed at 300 r/min and aeration rate at 3 L/min in a 5 L fermenter, the initial addition amount of konjac flour was 2%（w/v）, the initial pH of medium was 8.0, and the inoculum concentration was 6.7%（v/v）. Three batch fermentation kinetic models were established （cell growth kinetic model, substrate consumption kinetic model, product formation kinetic model） bases on Logistic and Luedeking-Piret equations. To be specific, cell growth kinetic model was dX/dt =0.431X （1- X/ 15.522 ）, substrate consumption kinetic model was -ds/dt =1.11 dX/dt ＋0.000 2 dP/dt ＋0.000 8X, and product formation kinetic model was dP/dt=133.1 dX ＋222.87X. The correlation coefficients R^2 of the three equations were 0.990 21, 0.989 08 and 0.988 12, respectively, which indicated a good correlation between experimental values and models. Therefore, the three equations could be used to describe the processes of cell growth, enzyme synthesis and substrate consumption during batch fermentation using B. licheniformis strain HDYM-04. The establishment of batch fermentation kinetic models （cell growth kinetic model, substrate depletion kinetic model, product formation kinetic model） could lay the theoretical foundation and provide practical reference for the applica- tion of HDYM-04 in fermentation industry.

Microbial aerosol characteristics in highly polluted and nearpristine environments featuring different climatic conditions

There is an increasing interest in understanding ambient bioaerosols due to their roles both in health and in climate. Here, we deployed an Ultraviolet Aerodynamic Particle Sizer to monitor viable （fluorescent） bioaerosol concentration levels at city centers （highly polluted） and their corresponding suburbs （near pristine） （total 40 locations） in 11 provinces featuring different climate zones in China between July 16 and 28, 2013. The concentration levels of viable bioaerosol particles （BioPM） of 〉0.5 μm were measured, and corresponding percentages of BioPM% （biological fraction of total PM） and BioPM2.5% （biological fraction of PM2.5） in particulate matter （PM） and BioPM, respectively, were determined. For some key cities, indoor viable bioaerosol levels were also obtained. In addition, bacterial structures of the air samples collected across these monitoring locations were studied using pyrosequencing. BioPM concentration levels ranged from 2.1 ×10^4 to 2.4 × 10^5/m3 for city centers [BioPM% = 6.4 % （4-6.3 %）] and 0.5 × 10^4 to 4.7 × 10^5/m3 for suburbs [BioPM% = 10 % （4-8.7 %）]. Distinctive bioaerosol size distribution patterns were observed for different climate zones, e.g., some had fluorescence peaks at 3 μm, while the majority had peaks at 1 μm. Ambient bacterial aerosol community structures were also found different for different geophysical locations. Results suggest that there was a poor overall relationship between PM and BioPM across 40 monitoring locations （R2= 0.081, two-tailed P value = 0.07435）. Generally, city centers had higher PM concentrations than suburbs, but not BioPM and BioPM%. Indoor bioaerosol levels were found at least tenfold higher than those corresponding outdoors. Bacillus was observed to dominate the bacterial aerosol community in the air sample.