Isolation from coastal sea water and characterization of bacterial strains involved in non-ionic surfactant degradation

A bacterial community degrading branched alkylphenol ethoxylate (APE) was selected from coastal sea water intermittently polluted by urban sewage. This community degraded more than 99% of a standard surfactant, TRITON X 100, but I.R. analysis of the remaining compound showed the accumulation of APE₂ (alkylphenol with a two units length ethoxylated chain) which seemed very recalcitrant to further biodegradation. Twenty-five strains were isolated from this community, essentially Gram negative and were related to Pseudomonas, Oceanospirillum or Deleya genera. Among these strains, only four were able to degrade APE_9–10 (TRITON X 100). They were related to the Pseudomonas genus and were of marine origin. Pure cultures performed with these strains on TRITON X 100 gave APE₅ and APE₄ as end products. These products were further degraded to APE2 by two other strains unable to degrade the initial surfactant.     

Isolation and characterization of surfactant degrading bacteria in a marine environment

Abstract Sea water and sediment samples taken near the coasts of Hyeres bay (France) were used for anionic surfactant titrations with surface and bottom waters and the finest part of sediments. The capacity for surfactant degradation by the 'in situ' microflora was evaluated. By using a selective plating technique 26 strains able to utilize anionic surfactant were isolated from the selected bacterial communities. Their ability to degrade anionic surfactant was verified according to the biodegradation standard method. Isolated strains were characterized by morphological and physiological properties using API 20 NE micro-method. All tested strains were Gram negative, strictly aerobic, rod or helical shaped. Their weak utilization of phenolic substrates suggests that they degrade preferentially the alkyl chain of the surfactant molecule. Biodegradation was more efficient with bacterial communities rather than with any isolated strains. Such observations indicate that complete mineralization involves several other so far non-isolated strains which complete the degradation initiated by the isolated strains.     

Effect of agitation rate on biomass and protease production by a marine bacterium Vibrio harveyi cultured in a fermentor

A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell-based medium, supplemented with skim milk under different hydrodynamic conditions, namely agitation and aeration rates. The addition of skim milk to Zobell medium enhanced fivefold the extracellular enzyme production. Protease production seemed to take place after maximum luminescence had been produced. Specific growth rate increased as a consequence of increasing agitation rates. The maximum activity of 4.28 units mg^–1 protein were formed with 700 rev min^–1 and 0.5 v/v/m. Protease activity detected has a molecular weight of 34 kDa. Another minor band of protease activity was found at 40 kDa.     

表面活性剂对嗜盐嗜碱硫碱弧菌D301生成及利用硫颗粒的影响

【背景】微生物脱硫是脱除气体中硫化氢的一种有效方法,其中,硫颗粒的生成与代谢是控制生物脱硫效率的关键,但目前相应的控制方法很少。【目的】研究不同种类表面活性剂对硫碱弧菌D301生成及利用硫颗粒的影响。【方法】通过摇床培养,利用X射线衍射、冷场发射扫描电镜、能谱分析及傅里叶红外光谱对硫颗粒进行表征。【结果】单质硫主要以S8形式存在,吐温-80和十二烷基磺酸钠(Sodium Dodecyl Sulfate,SDS)的添加对硫颗粒的形态及生成量影响明显。对照组中生成的硫颗粒呈规则球形,光滑完整,其表面附着蛋白质等生物大分子;加入0.01 g/L吐温-80后,硫颗粒呈长杆状、颗粒增大、利用速率减慢;加入0.3g/L的SDS后,硫颗粒呈短棒状、生成量减少、利用速率加快,同时延缓了硫碱弧菌D301的衰亡。【结论】添加表面活性剂可以改变硫颗粒形态并且影响其利用,是一种调控硫颗粒生成和代谢的有效手段。     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

Effect of a transient perturbation on marine bacterial communities with contrasting history

Aims: To evaluate the importance of the bacterial composition on the resilience of the organic matter assimilation in the sea. Methods and Results: Chemostats were inoculated with coastal and offshore bacterial communities. Bacterial density and protein synthesis increased before stabilizing, and this response to confinement was more marked in the offshore chemostats. Before the toluene perturbation the community structure in the coastal chemostats remained complex whereas the offshore chemostats became dominated by Alteromonas sp. After the perturbation, bacterial protein synthesis was inhibited before peaking briefly at a level fivefold to that observed before the perturbation and then stabilizing at a level comparable to that before the perturbation. Alteromonas dominated both the coastal and the offshore communities immediately after the perturbation and the coastal communities did not recover their initial complexity. Conclusions: Cell lysis induced by the toluene perturbation favoured the growth of Alteromonas which could initiate growth rapidly in response to the nutrient pulse. Despite their different community structure in situ, the resilience of protein synthesis of coastal and offshore bacterial communities was dependent on Alteromonas, which dominated in the chemostats. Significance and Impact of the Study: Here we show that although Alteromonas sp. dominated in artificial offshore and coastal communities in chemostats, their response time to the shock was different. This suggests that future perturbation studies on resilience in the marine environment should take account of ecosystem history.     

Effect of a commercial alcohol ethoxylate surfactant (C<Subscript>11-15</Subscript>E<Subscript>7</Subscript>) on

Biodegradation of poorly soluble polycyclic aromatic hydrocarbons (PAHs) has been a challenge in bioremediation. In recent years, surfactant-enhanced bioremediation of PAH contaminants has attracted great attention in research. In this study, biodegradation of phenanthrene as a model PAHs solubilized in saline micellar solutions of a biodegradable commercial alcohol ethoxylate nonionic surfactant was investigated. The critical micelle concentration (CMC) of the surfactant and its solubilization capacity for phenanthrene were examined in an artificial saline water medium, and a type of marine bacteria, Neptunomonas naphthovorans, was studied for the biodegradation of phenanthrene solubilized in the surfactant micellar solutions of the saline medium. It is found that the solubility of phenanthrene in the surfactant micellar solutions increased linearly with the surfactant concentrations, but, at a fixed phenanthrene concentration, the biodegradability of phenanthrene in the micellar solutions decreased with the increase of the surfactant concentrations. This was attributed to the reduced bioavailability of phenanthrene, due to its increased solubilization extent in the micellar phase and possibly lowered mass transfer rate from the micellar phase into the aqueous phase or into the bacterial cells. In addition, an inhibitory effect of the surfactant on the bacterial growth at high surfactant concentrations may also play a role. It is concluded that the surfactant largely enhanced the solubilization of phenanthrene in the saline water medium, but excess existence of the surfactant in the medium should be minimized or avoided for the biodegradation of phenanthrene by Neptunomonas naphthovorans.     

Emission of carbonyl sulfide by Thiobacillus thioparus grown with thiocyanate in pure and mixed cultures

Abstract The biological activity of the heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was found to be predominantly associated with the periplasmic extract (about four-fold higher than the culture supernatant) of a recombinant E. coli (JM109) strain carrying the NAG-St toxin gene. Four molecular species of NAG-ST, two each from the periplasmic extract and culture supernatant of JM109, were purified. Amino acid sequence analysis of the four NAG-ST peptides isolated by HPLC revealed that they all differed from that of the mature 17-amino acid residue NAG-ST released by V. cholerae non-O1. The M _r-values of the peptides obtained from the periplasmic extract were 4331 and 2785, while those recovered from the culture supernatant were 3154 and 2785. It thus appears that V. cholerae NAG-ST is synthesized as larger molecules in the recombinant E. coli strain. The differences in sizes of the exported NAG-ST molecule could relate to difference in the enzyme cleavage system between E. coli and V. cholerea .     

近岸海洋线虫与细菌、古菌的共现性及其对碳、氮循环的影响

【目的】初步探究海洋线虫与微生物的相互作用对碳、氮循环的影响。【方法】利用16S r RNA和18S r RNA基因高通量测序方法,对33个近岸沉积物样品中细菌、古菌和真核生物的多样性进行调查;对海洋线虫与细菌、海洋线虫与古菌的共现性进行网络分析,并采用Spearman统计学方法,识别出与海洋线虫共现性呈显著相关性的微生物种类。【结果】在夏季,红树林和潮间带泥滩样品中线虫OTU平均相对丰度基本呈随深度增加而递减趋势;冬季的红树林样品中发现相类似变化规律,只有在冬季潮间带泥滩样品中线虫OTU平均相对丰度在深层较高于表层。相对丰度最高的海洋线虫隶属于单宫目(47%)、色矛目(19%)、刺嘴目(16%)和垫刃目(9%),它们与热源体古菌、深古菌、γ-和δ-变形菌等微生物有显著正/负相关关系。【结论】在香港米埔湿地沉积物中,与相对丰度最高的5种线虫显著相关的几大类微生物均在碳、氮、硫等元素循环方面起十分重要的作用,暗示海洋线虫与微生物潜在的相互作用对元素地球化学循环具有重要影响。研究结果有助于深入了解线虫在生态系统中未被揭示的生态功能,有助于更清晰地认识海洋线虫在底栖生态系统中所扮演的角色。     

L-Arg对LPS诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响

目的:探讨L-精氨酸(L-Arg)对脂多糖(LPS)诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响。方法:舌下静脉注射脂多糖(LPS)复制肺损伤模型。健康雄性SD大鼠48只,随机分为对照组、模型组(LPS组)和L-Arg治疗组(L-Arg组)(n=16)。分别于给予LPS 3 h或6 h后给予生理盐水(对照组及LPS组,ip)和L-Arg(500 mg/kg ip)(L-Arg治疗组),治疗3 h。原位杂交法(ISH)检测肺组织中肺表面活性蛋白A(SP-A)mRNA的表达;测定肺泡灌洗液(BALF)中的总蛋白(TP)。体外分离培养大鼠肺泡巨噬细胞,以LPS(终浓度10 mg/L)处理巨噬细胞,观察L-Arg对肺泡巨噬细胞的影响。结果:与对照组比较,大鼠肺损伤后SP-A mRNA表达减弱,BALF中TP增多(P<0.01)。肺损伤3 h用L-Arg治疗3 h后,SP-A mRNA阳性细胞表达明显增强,BALF中TP较LPS组相同时间点明显降低(P<0.05,P<0.01),肺损伤减轻。体外实验中,与正常对照组相比,LPS组细胞培养上清中乳酸脱氢酶(LDH)、一氧化氮(NO)、肿瘤坏死因子-α(TNFα-)和白细胞介素-6(IL-6)浓度明显增高(P<0.01);L-Arg明显减少LPS所致的LDH的释放,降低TNFα-和IL-6浓度。结论:L-Arg可减轻内毒素性肺损伤,此机制可能与增强SP-AmRNA表达有关;LPS可刺激巨噬细胞分泌促炎因子和NO,L-Arg可抑制LPS对巨噬细胞的作用。     

海水中主要金属盐对4株Kangiella属细菌的生长及其蛋白酶相关基因表达的影响

【背景】Kangiella是海洋专属的、营异养生长的革兰氏阴性菌。按16S rRNA基因系统发育分析,Kangiella属归属于γ-变形杆菌纲,但此类细菌的生理生态功能未知。推测该类海洋细菌产生的胞外丝氨酸蛋白酶在其利用有机氮源生长的过程中起重要作用。【目的】研究海水盐中主要金属离子Na盐、Mg盐、Ca盐、K盐对海洋来源的4株Kangiella菌株(K.aquimarina DSM16071、K.geojedonensis YCS-5、K.koreensis DSM 16069和K.profundi FT102)的生长及胞外蛋白酶表达的影响。【方法】测定Kangiella属4个不同种的模式菌株在2216E培养基及不添加Na盐、Mg盐、Ca盐和K盐的2216E培养基中的生长情况。以2%的酪蛋白为底物,采用福林酚法分别检测4株菌株的胞外蛋白酶酶活。克隆K.profundi FT102菌株中的3个丝氨酸蛋白酶编码基因以及phoP和phoQ基因,使用实时荧光定量PCR技术检测Mg盐对这些基因表达的影响。【结果】4株Kangiella菌株在不添加Na盐的2216E培养基中均不能生长;在不添加Mg盐、Ca盐和K盐的2216E培养基中,K.aquimarina DSM 16071、K.geojedonensis YCS-5和K.profundi FT102这3个菌株的生长差异较小;而K.koreensis DSM 16069菌株的生物量在不添加上述3类主要金属盐的2216E培养基中反而增高。各个菌株在对数期时的胞外蛋白酶活差异较大,最高可以达到11.23 U/mL,最低仅为0.99 U/mL。2216E培养基中不添加Mg盐时,K.aquimarina DSM 16071、K.geojedonensis YCS-5和K.profundi FT102这3株菌中的丝氨酸蛋白酶编码基因asp1、asp2和asp3以及phoP和phoQ基因的转录水平无显著差异,而在K.koreensis DSM 16069菌株中这5个基因的表达却显著上调。【结论】Na盐是4株海洋Kangiella菌株生长所必需的金属离子,Mg盐和Ca盐对4株菌株的生长及其中3个保守的丝氨酸蛋白酶基因表达的影响不同。在不添加Mg盐的2216E培养基中,K.koreensis DSM 16069菌株的生物量、胞外蛋白酶活和3个保守的丝氨酸蛋白酶编码基因以及phoP和phoQ基因的转录量都显著上升。     

Optimization and modeling of phenanthrene degradation by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. 6PY1 in a biphasic medium using response-surface methodology

In the present paper, the degradation of phenanthrene, a model polycyclic aromatic hydrocarbon compound, by the Mycobacterium strain 6PY1 was optimized in a biphasic culture medium. The optimization and modeling were performed using the design of experiments methodology. The temperature, the silicone oil/mineral salts medium volume ratio, and the initial cell concentration, were used as the central composite design parameters. In all experiments, the phenanthrene was degraded to undetectable levels. Response surface methodology was successfully employed to derive an empirical model describing the rate and time of degradation and to deduce the optimal degradation conditions. As a result of the optimization processes, the optimal responses for the degradation rate, the volumetric degradation rate, and the 90% degradation time were estimated to be 0.172 mg h⁻¹, 22 mg l⁻¹ h⁻¹, and 18 h, respectively.     

Effects of inorganic nutrient levels on the biodegradation of benzene, toluene, and xylene (BTX) by Pseudomonas spp. in a laboratory porous media sand aquifer model

The effect of inorganic nutrients (sulfate, phosphate, and ammonium chloride) on the aerobic biodegradation of benzene, toluene, and xylene (BTX) by Pseudomonas spp. was studied in the laboratory using a glass sand tank. The increase of nutrient levels resulted in enhanced bacterial growth and BTX degradation. Sulfate and phosphate serve as key electron acceptors in the microbiological processes degrading BTX. The observed bacterial morphological changes during BTX degradation reveal that the filamentous bacteria were the dominant species at low temperatures about 20 degrees C. The spherical and rod-shaped cells became dominant at higher temperatures ranging from 25 degrees C to 28 degrees C. When the BTX mixture was allowed to be biodegraded for longer incubation periods of 21-42 h at high phosphate concentrations, large amounts of rod-shaped cells were clustered. The morphological adaptation appears to be controlled by the temperature and nutrient levels in the sandy medium where Pseudomonas spp. thrives.     

Effect of aroclor 1248 and two pure PCB congeners on phospholipase D activity in rat renal tubular cell cultures

This paper elucidates the effect of different polychlorinated biphenyls (PCBs) on the phospholipase D (PLD) activity in soluble and particulate fractions of rat renal proximal tubular culture cells. Treatment with Aroclor 1248 (a commercial PCB mixture) caused a marked increase in the activity of PLD in intact renal tubular cells. The PLD activity was increased by Aroclor 1248 in the particulate fraction while the enzyme activity was unaffected in the soluble fraction. This work also shows that PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) can increase the activity of PLD only in the particulate fraction. The exposure of cell cultures to PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener) does not alter PLD activity. These results suggest that PCB effects are structure dependent. Therefore, in order to clarify the molecular mechanism of activation of PLD by PCBs, the contents of immunoreactive PLD were examined by immunoblot analysis. Renal tubular cells expressed a PLD protein of 120 kDa corresponding with the PLD1 mammalian isoform in both the particulate and the soluble fraction. Aroclor 1248, PCB 153, and PCB 77 do not induce changes in the levels of PLD protein. These data indicate that PCBs, particularly nonplanar congeners, increase PLD activity. Moreover, these changes could not be demonstrated in the enzyme content in rat renal tubular cell cultures.     

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.     

Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp.

A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo[a]pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor. Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded. Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.     

Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor

This article studies the operation of a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) at different applied organic load rates (OLRs). The process is based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. A mixture of acetic, lactic, and propionic acids at different concentrations (in the range 8.5-31.25 gCOD/L) was fed every 2 h in a sequencing batch reactor (SBR). The resulting applied OLR was in the range 8.5-31.25 gCOD/L/day. Even though, as expected, the increase in the OLR caused an increase in biomass concentration (up to about 8.7 g COD/L), it also caused a relevant decrease of maximal polymer production rate. This decrease in polymer production rate was related to the different extent of "feast and famine" conditions, as function of the applied OLR and of the start-up conditions. As a consequence the best performance of the process was obtained at an intermediate OLR (20 gCOD/L/day) where both biomass productivity and PHA storage were high enough. However, at this high OLR the process was unstable and sudden decrease of performance was also observed. The sludge characterized by the highest PHA storage response was investigated by 16S rDNA clone library. The clone library contained sequences mostly from PHA producers (e.g., Alcaligenes and Comamonas genera); however many genera and among them, one of the dominant (Thauera), were never described before in relation to PHA storage response.     

补骨脂素对TCP磨损颗粒所致大鼠成骨细胞损伤的干预作用及其机制

目的: 探讨补骨脂素(psoralen)对TCP磨损颗粒诱导成骨细胞损伤的影响及其分子机制。方法: 通过消化法从SD大鼠乳鼠颅骨中获取原代的成骨细胞,应用碱性磷酸酶(ALP)染色鉴定成骨细胞。TCP磨损颗粒(0.1 mg/ml)与成骨细胞共孵育48 h构建成骨细胞损伤的体外实验模型,实验随机分为正常对照组(Control)、模型(TCP)组和psoralen(10^-7 mol/L、10^-6 mol/L和10^-5 mol/L)组。WST法和流式细胞术分别检测各组成骨细胞活性变化和凋亡情况;化学比色法检测成骨细胞中ALP活性;各组成骨细胞培养14 d后应用茜素红S染色观察矿化结节形成。Western blot法检测各组成骨细胞中葡萄糖调节蛋白78/94(GRP78/94)、肌醇依赖酶1α(IRE1α)、剪切型X盒结合蛋白1(XBP1s)和磷酸化c-Jun氨基末端激酶(p-JNK)等蛋白质的表达。结果: 与Control组比较,TCP组成骨细胞活性、ALP活性和矿化结节的生成显著降低(P＜0.05),细胞凋亡率、GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达明显增加(P＜0.05);与TCP组比较,补骨脂素各组成骨细胞损伤情况明显减轻,细胞凋亡率显著减少(P＜0.05),GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达也明显下降(P＜0.05)。结论: 补骨脂素可抑制TCP磨损颗粒诱导的IRE1α-XBP1s-JNK信号通路的激活,阻止TCP颗粒所致的成骨细胞损伤及凋亡。