Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.

Demineralization of Gondwana coal with Pseudomonas mendocina strain B6-1： a case study of coal from Gopinathpur top and bottom seams of Mugma mine, Dhanbad, Jharkhand （India）

In the present investigation an attempt has been made to demineralize the Gondwana coal of Gopinathpur top and bottom seams of Mugma mine, Raniganj coalfield, Dhanbad with the help of Pseudomonas mendocina strain B6-1. The change in the amount of ash yield and decrease in the concentration of selected minor elements like Na, K, Mn and Ca and environmentally sensitive selected trace elements such as Cd, Pb, Se, Ni, Mn, and Zn have been studied as a function of time of bacterial treatment as well as with variation in the bacterial biomass. After 28 days of bacterial treatment there was variable amount of decrease observed in ash yield as well as in the concentration of minor and trace elements. The removal of the elements was further enhanced with the increase in the bacterial biomass from 10 to 25 mg/mL. Due to over exploitation of superior grade coals in the country, the remaining coal resources, available for current use, are inferior in grade and contain high level of impurities and there is ample scope of bio-beneficiation of these coals using bacterial biomass.

Pseudomonas mendocina LYX:A novel aerobic bacterium with advantage of removing nitrate high effectively by assimilation and dissimilation simultaneously

The problem of nitrate accumulation in aerobic tank and total nitrogen excessive discharge in effluent was very common in traditional livestock and poultry farming wastewater treatment systems owing to the lengthy process flow and low process control level.A strain LYX of aerobic bacterium was isolated from the activated sludge of a wastewater treatment system in a pig farm,which could remove nitrate effectively in aerobic tank and was identified Pseudomonas mendocina by 16S rRNA sequencing.Under the condition of nitrate as the sole nitrogen source,this strain removed over 90%of NO_(3)^(−)-N with an initial concentration of 110 mg/L under aerobic conditions within 48 hours.Among them,37.9%of NO_(3)^(−)-N was assimilated into Bio-N,about 51.9%was reduced to gaseous nitrogen and less than 0.5%of nitrogen was replaced by NO_(3)^(−)-N and NH_(4)^(+)-N,9.7%NO_(3)^(−)-N remained in the effluent at the end.At the same time,four key genes(napA,nirK,norB and nosZ)related to nitrate nitrogen removal were expressed during the denitrification process of P.mendocina LYX,in which the transcription level of the indicator genes of this aerobic denitrifying bacterium(napA)was the highest.In addition,it was found with the 15N tracer technique that inoculation of this strain on sludge increased the amount of nitrogen loss from 9.26 nmol N/(g·h)to 23.835 nmol N/(g·h).Therefore,P.medocina LYX is a potential bioagent for advanced nitrogen removal by assimilating and reducing nitrate simultaneously in aerobic tanks.

过表达Pseudomonas mendocina复制起始相关蛋白提高菌体繁殖速度

复制起始蛋白DnaA通过识别复制起始位点oriC上的特殊序列,并招募复制复合体起始复制,它通过绑定ATP或ADP来对复制起始的开始进行调节.DNA聚合酶Ⅲ的β亚基DnaN蛋白,负责将DNA聚合酶III连接到已经解旋的DNA上,以开始复制过程.通过过表达复制起始相关蛋白DnaA和DnaN,促进了菌体的繁殖能力,优化了菌体的生长状态,缩短了菌体倍增时间,为构建Pseudomonas mendocina工程菌株提供了参考.

Production of Medium Chain Length Polyhydroxyalkanoates by Pseudomonas mendocina 0806 from Related and Unrelated Carbon Sources

Pseudomonas mendocina strain 0806 isolated from oil\|contaminated soil was found to produce medium chain length polyhydroxyalkanoates (mcl PHAs). The mcl PHAs consist of monomers with even numbers of carbon atoms such as hydroxyhexanoate (HHx or C6), hydroxyoctanoate (HO or C8), and/or hydroxydecanoate (HD or C10) as major compositions when the strain was grown on unrelated carbon sources such as glucose, citric acid and related carbon sources such as octanoate, myristic acid or oleic acid. While even and odd number hydroxyalkanoate (HA) monomers were synthesized when tridecanoic acid was used as carbon source. The molar ratio of carbon to nitrogen ( R C/N ) had strong effects on PHA compositions: the strain produced PHAs with 97%-99% (molar ratio) HD (C10) monomer when grown in a glucose ammonium sulfate medium of R C/N <20; while 20% HO and 80% HD (molar fraction) were found as compositions in the polymers when growth was conducted in the media containing R C/N >40. It was demonstrated that the molar ratio of HO/HD remained constant in the polymers synthesized from media containing a constant R C/N , regardless of the change of glucose concentration. Up to 3 6 g/L cell dry weight containing 45% (mass fraction) PHAs was produced by the strain grown for 48?h in a medium containing 25?g/L glucose with R C/N of 40.

固定化好氧脱氮菌及其在低C/N鸭场废水处理中的应用

为解决集约化畜禽养殖废水处理工艺(厌氧-缺氧好氧-缺氧好氧)中普遍存在的出水NO 3--N浓度超标的问题,试验将一株具有高效去除NO 3--N能力的好氧细菌Pseudomonas mendocina LYX固定于丝瓜络,并制备成菌剂,研究其在最佳脱氮条件下对鸭场废水深度处理的强化效果.结果表明:①在C N为5、培养温度为35℃、pH为8、ρ(DO)为3.45 mg L的条件下,P.mendocina LYX达到最优脱氮效果.②固定化菌剂投加后,反应器中NO 3--N和COD Cr的去除率较菌剂强化前分别提高了40%和20%,ρ(NO 2--N)提前达到峰值,反硝化进程被明显推进.③反应器运行至第17天时,污泥中好氧脱氮菌Pseudomonas的相对丰度由最初的0.03%升至8.50%,反应器内微生物多样性显著增加,此时,NO 3--N和COD Cr去除率均达到最大值,分别为86%和79%.④反应器运行至第22天时,污泥中Pseudomonas的相对丰度降至0.59%,菌剂强化作用逐渐减弱,反应器中NO 3--N和COD Cr去除率基本与菌剂强化前持平,菌剂强化效果具有周期性.研究显示,固定化P.mendocina LYX菌剂有望在好氧条件下通过原位强化解决低C/N水质中的NO 3--N污染问题,具有较好的应用前景.

Isolation and Characterization of Microorganisms for Degradation of Minimal Oils and Fats at Low Temperatures

The isolation of microorganisms for biodegradation of minimal fats and oils at low temperatures was reported. By using rapeseed oil as a sole carbon source, six strains were isolated from five kinds of oils/fats-contaminated wastewater, soil, and active sludge. Among them, two strains which show the highest oil removal ratios were identified as Pseudomonas pseudoalcaligenes and Pseudomonas mendocina, respectively. The experiments of orthogonal impact conditions show that the optimal oil degradation condition is at pH 8.0, 5 ℃ and 100 mg/L oil. Under this condition, the rapeseed oil degradation ratios of two strains after 24 hours amount to 92.6% and 92.0% respectively, whereas the removal ratios of lard decrease to 39.5% and 54.3%.

Nitric oxide removal by wastewater bacteria in a biotrickling filter

Nitric oxide （NO） is one of the most important air pollutants in atmosphere mainly emitted from combustion source. A biotriclding filter was designed and operated to remove NO from an air stream using bacteria extracted from the sewage sludge of a municipal sewage treatment plant. To obtain the best operation conditions for the biotrickling filter, orthogonal experiments （L9（34）） were designed. Inlet oxygen concentration was found to be the most significant factor of the biotrickling filter and has a significant negative effect on the system. The optimal conditions of the biotrickling filter occurred at a temperature of 40℃, a pH of 8.0 and a chemical oxygen demand of 165 mg/L in the recycled water with no oxygen in the system. The bacteria sample was detected by DNA sequencing technology and showed 93%-98% similarity to Pseudomonas mendocina. Moreover, a full gene sequencing results indicated the bacterium was a brand new strain and named as P. mendocina DLHK. This strain can transfer nitrate to organic nitrogen. The result suggested the assimilation nitrogen process in this system. Through the isotope experimental analysis, two intermediate products （^15NO and ^15N2O） were found. The results indicated the denitrification function and capability of the biotrickling filter in removing NO.