利用有益微生物改善养殖生态环境的研究

研究了噬菌蛭弧菌在实验室条件下对水产养殖中常见病菌的裂解能力及噬菌蛭弧菌和光合细菌对池塘生态环境的改善作用。结果表明 ,噬菌蛭菌对嗜水气单胞菌具有裂解作用 ,噬菌蛭弧菌和光合细菌结合使用对养殖环境具有明显的改善作用。 2 5d后实验组比对照组池塘中的细菌总数少 2个数量级 ,70d后实验组比对照组池塘中的细菌总数少 3个数量级 ;2 5d后对照组COD为 6 35mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的COD分别为 4 6 7、4 1 6、4 1 8mg L ;2 5d后对照组NH3 N为 0 4 9mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的NH3 N分别为 0 36、0 33、0 32mg L ,90d后对照组NH3 N为 0 4 1mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的NH3 N分别为 0 2 7、0 2 2、0 2 1mg L ;5 5d后对照组硫化物为0 0 1 1mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的硫化物分别为0 0 1 0、0 0 0 8、0 0 0 8mg L。     

噬菌弧菌N1对淡水和海水养殖水体细菌群落影响PCR-DGGE分析

【目的】研究噬菌弧菌Bacteriovorax sp. N1在一个投放周期内对淡水和海水养殖环境中细菌、弧菌总数及细菌群落结构的影响。【方法】自市售微生态制剂分离蛭弧菌N1并进行分子鉴定,测定其裂解效果,制备高浓度N1菌液分别投放至淡水红鲤鱼(red carp)和海水仿刺参(Apostichopus japonicus)养殖水体,采用细菌平板计数法及PCR-变性梯度凝胶电泳(PCR-DGGE)技术分析48 h内水体环境中细菌、弧菌总数及细菌群落结构变化。【结果】经鉴定蛭弧菌N1为噬菌弧菌,其对大肠杆菌(Escherichia coli)、溶藻弧菌(Vibrio alginolyticus)、黄海希瓦氏菌(Shewanella smarisflavi)、灿烂弧菌(Vibrio splendidus)、哈氏弧菌(Vibrio harveyi)和金黄色葡萄球菌(Staphylococcus aureus)均有裂解效果。将噬菌弧菌N1投放进淡水和海水养殖环境的12～24 h,其能显著降低两种环境中弧菌含量(P 0.05)。DGGE分析显示添加噬菌弧菌N1后淡水组优势菌群弧菌属(Vibrio,a1)和不可培养杆菌属(Uncultured bacterium, a5)在12 h以后含量有明显的减少,假单胞菌属(Pseudomonas, a3)和红杆菌科Shimia属(Shimia, a6)菌含量增加。海水组优势菌群不可培养杆菌属(c2)菌在12 h时变成非优势菌群,而白杆菌属(Albirhodobacter,c1)增加成为优势菌群。噬菌弧菌N1在水中含量在24 h时降至最低。【结论】噬菌弧菌N1对海水和淡水环境中的弧菌属和不可培养杆菌属菌群有明显的裂解作用导致其含量下降,但也使假单胞菌属(Pseudomonas),红杆菌科Shimia属和白杆菌属(Albirhodobacter)菌群含量增加,但生物效应不明。为维持噬菌弧菌N1对弧菌的控制,需要24 h左右重新补充投放。     

利用有益微生物改善养殖生态环境的研究

研究了噬菌蛭弧菌在实验室条件下对水产养殖中常见病菌的裂解能力及噬蛭弧菌的光合细菌对池塘生态环境的改善作用。结果表明，噬菌蛭菌对嗜水气单胞菌具有裂解作用，噬菌蛭弧菌和光合细菌结合使用对养殖环境具有明显的改善作用。25d实验组比对照组池塘中的细菌总数少2个数量级，70d后实验组比对照组池塘中的细菌总数少3个数量级，25d对照组COD为6．35mg/L，使用噬菌蛭 菌浓度为3，5，10mL/m^3及光合细菌皆为5mL/m^3实验组的COD分别为4．67，4．16，4．18mg/L；25d后对照组NH3－N为0．49mg/L，使用噬菌蛭弧菌浓度为3，5,10mL.m^3及光合细菌皆为5mL/m^3实验组的NH3－N分别为0．36，0．33，0．32mg/L，90d后对照组NH3－N为0．41mg/L，使用噬菌蛭弧菌浓度为3，5，10mL/m^3，及光合细菌皆为5mL/m^3实验组的NH3－N分别为0．27，0．22，0．21mg/L,55d后对照组硫化的为0．011mg/L，使用噬菌蛭弧菌浓度为3，5，10mL/m^3及光合细菌皆为5mL/m^3实验组的硫化物分别为0．010，0．008，0．008mg/L。     

不同养殖模式虾池弧菌对抗菌药物的耐药性与虾池水质评价

研究了湛江东海岛3种不同养殖模式虾池弧菌对抗菌药物的耐药性,并对虾池水域环境的水温、盐度、DO、pH、叶绿素a、DOC、无机氮、无机磷、硅酸盐、细菌总数和弧菌数量变化进行了监测和评价。结果表明,调查的三个虾池的弧菌对万古霉素产生耐药性,高位新池和传统精养池的弧菌对利福平产生耐药性,对其他抗菌药尚未形成耐性,3个虾池的弧菌对恩诺沙星和氯霉素高度敏感,传统精养池和天然虾池的弧菌对复方新诺明、庆大霉素、TMP和环丙沙星高度敏感;在水平差异上,高位新池的弧菌比传统精养池和天然虾池对12种抗菌药更具耐药性,同时多重耐药菌株在高位新池中出现,可能与虾苗来源和配合饲料中添加了抗菌药物有关。水质监测结果表明,养殖过程中,虾池水域环境逐渐富营养化,无机氮和Chla超过富营养化阈值,细菌总数增加;两个精养池的水体富营养化程度高于天然虾池;调查期间的弧菌数量在对虾发病的感染剂量阈值范围之内,尚无弧菌病害发生的潜在危险。     

马氏珠母贝肠道及其养殖水体可培养细菌群落结构

【目的】了解马氏珠母贝(Pinctada fucata martensii)肠道及其养殖水体可培养细菌的群落组成。【方法】采用2216E平板涂布法研究海区养殖马氏珠母贝肠道与养殖水体的可培养菌群种类及丰度。【结果与结论】马氏珠母贝肠道及其养殖水体的可培养细菌归属于2门(变形菌门和厚壁菌门)3纲7目10科23属56种。属水平上,肠道中以弧菌属(74.7%)和假交替单胞菌属(18.7%)为主;养殖水体中α-变形菌纲的FJ943236_g属(40.7%)丰度最大,弧菌属(16.7%)相对肠道丰度较低。样品共有菌属为弧菌属、假交替单胞菌属、发光杆菌属和芽孢杆菌属;肠道特有菌属为希瓦氏菌属和盐单胞菌属;养殖水体特有菌属主要为FJ943236_g、鲁杰氏菌属和Nautella。在种水平上,7个种为二者共有;马氏珠母贝肠道和养殖水体特异性菌种分别为18个和31个。虽然门水平上马氏珠母贝肠道中可培养细菌群落与其养殖水体中的细菌群落大致相似,但在属、种水平上二者差异明显。     

有效微生物菌群对养虾水体细菌生态和水质的影响

研究有效微生物菌群(EM)对养虾水体中异养细菌、弧菌、光合细菌、放线菌的数量以及水体水质的影响。结果表明,在养殖池中添加EM,对水体中的异养细菌、弧菌、光合细菌及放线菌的数量均有显著影响:实验组异养细菌数量比传统对照池减少40.0%,弧菌数量减少7.8%,光合细菌数量增加98.2%,放线菌数量增加99.2%。EM能改良水质条件,与对照组相比,实验组溶解氧提高11.0%,COD降低8.0%,氨氮含量降低20.7%,亚硝酸氮含量降低10.0%。另外,实验组对虾成活率比对照组提高23.08%,饵料系数下降9.4%,利润提高了16.11%。     

九孔鲍养殖水体中产酶菌群的研究

为了解九孔鲍养殖环境中细菌生理菌群的生态作用,对分离自养殖水体的33株菌株的产酶能力和种类进行了研究。结果表明,具有分泌胞外蛋白酶、脂肪酶和纤维素酶能力的菌株分别为39.4%、57.6%和30.3%;在这33株菌中,产至少一种胞外酶的菌株比例高达63.6%。API鉴定结果表明,在有能力分泌胞外蛋白酶、脂肪酶和纤维素酶的菌株中,弧菌菌群所占的比例分别为46%、42%和40%,而其中河流弧菌又占各弧菌菌群比例的83.3%、62.5%和100%。充分阐明弧菌,尤其是河流弧菌为优势菌,在鲍鱼养殖微生物态中起的重要作用。     

大亚湾网箱养殖区异养细菌和弧菌的数量动态

研究了大亚湾网箱养殖水体中细菌的数量动态,以探讨其变化规律与鱼病的关系。结果表明:总细菌、弧菌的数量变化各异,网箱内、网箱外和对照海区细菌数量分布与变化不同。养殖区异养细菌丰值期在4-5月份,5月份最高;网箱水体弧菌的丰值期在6-11月,最高在8月;网箱外丰值期在7-9月,最高在8月,对照海区最高值在9月,其他时间变化不大。无论是异养菌还是弧菌,细菌值大小顺序为网箱内、网箱外、对照海区。弧菌数量与部分环境因子相关性较为密切,但相关程度随不同空间而不同,可能受其他环境条件影响。弧菌的高值期与弧菌病的流行期相吻合,鱼体受伤或其他原因引起细菌感染也会使水体中细菌数量升高。     

生物滤料对翘嘴红鲌生长、水质及细菌变化的影响

在实验池投放一定数量的生物刷和生物球等生物滤料,研究其对翘嘴红鲌生长、池塘水质及细菌变化等的影响。结果表明:与对照池相比,实验池翘嘴红鲌生长有增加趋势,对池塘水温、pH、DO、TP、H2S等指标无显著影响(P>0.05),但是在30 d时水体COD显著降低(P<0.05);在90 d时NH4-N显著降低(P<0.05);在60 d和90 d时NO2-N显著降低(P<0.05);与对照池相比,在30 d时实验池水样与生物球中异养菌的数量显著增加(P<0.05);在30、60 d时,实验池生物刷中异养菌的数量显著增加(P<0.05);在90 d时实验池水样、生物球、生物刷氨化细菌、反硝化细菌的数量显著增加(P<0.05);实验池水样、生物球分别在30、90 d时弧菌数量显著降低(P<0.05)。表明生物滤料可增加池塘的异养菌、氨化细菌、反硝化细菌数量,降低水体弧菌数量,一定程度上改善养殖水体,促进鱼体生长。     

广东流沙湾网箱养鱼区水体氮磷的时空变化及负荷估算

研究流沙湾网箱养鱼区水体氮、磷含量的月变化和水平分布,并运用质量平衡方程估算其氮、磷负荷。结果表明:总氮变化在0.212~0.892 mg/L之间,平均0.498 mg/L;总磷变化在0.009~0.036 mg/L之间,平均0.020mg/L;氮磷比变化在23.6~26.1之间,平均为25。在5-9月,氮、磷含量变化呈逐渐增加趋势,到9月达到最大,10月又减少。网箱内部氮、磷的含量高于网箱外部,并随着与网箱距离的增大而逐渐减小。红鳍笛鲷(Lutjanus argentimaculatus)、紫红笛鲷(Trachinotus ovatus)和卵形鲳鲹(Lutjanus erythopterus)全鱼的氮质量分数平均为2.42%,磷质量分数平均为0.26%,饵料系数为6.67,流沙湾网箱养鱼养殖期产生的氮、磷负荷平均分别为135.8和19.4 kg/t。     

Biodegradation of Enteromorpha Polysaccharides by Intestinal Micro-Community from Siganus oramin

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides(EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U m L~(-1) in 32 hours. With denaturing gradient gel electrophoresis(DGGE) and 16 S r RNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro-community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

红鳍笛鲷弧菌病病原的研究

从广东湛江特呈岛鱼排取患病红鳍笛鲷进行病原分离、纯化,得到一株病原菌Ls 1。该菌 为革兰阴性短杆菌,有运动性,菌落半透明,最适温度28～30℃,需盐生长,氧化酶反应阳性,发酵 葡萄糖不产气,不发酵肌醇,对弧菌抑制剂O/129敏感。经API ID32E细菌鉴定系统及弧菌科细 菌生化鉴定管鉴定,该菌为一株溶藻弧菌(Vibrioaginolyticus);其对红鳍笛鲷的半数致死量为6.32 ×105mL-1。药敏试验结果表明,该菌对阿莫西林、先锋V不敏感,对新生霉素、卡那霉素、多粘菌 素B、四环素中度敏感,而对庆大霉素、氟哌酸、氯霉素、红霉素、链霉素和复方新诺明等有较高的敏 感性。     

石油污染物的微生物降解

从炼油厂污水池底泥中富集、驯化、分离、筛选,得到4种优势石油降解菌。采用摇床培养,研究了各优势菌和混合菌对石油烃的降解性能。结果表明:4种菌和混合菌20 d可将初始质量浓度为10000 mg/L的石油烃依次降解90.8%、88.9%、57.8%、49.8%、91.2%;培养液中石油烃的半衰期依次为5.5、6、15、19、5 d。初步鉴定4种菌分别属:节细菌(Arthrobacter sp.)、芽胞杆菌(Bacillus sp.)、不动杆菌(Acinetobacter sp.)、不动杆菌(Acinetobacter sp.)。     

Optimization of culturing condition and medium composition for the production of alginate lyase by a marine <Emphasis Type="Italic">Vibrio</Emphasis> sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.     

复方中草药对吉富罗非鱼生长及肠道菌群的影响

将A、B、C三种复方中草药分别按质量分数1.5%的比例添加到罗非鱼商品饲料中,喂养初始体重约16.58±0.48g的吉富罗非罗28d,通过测定罗非鱼的增重率、饲料系数、肠道菌群等指标,研究三种复方中草药制剂对罗非鱼生长性能及肠道菌群的影响。结果显示:三种复方中草药制剂对罗非鱼的增重率、成活率、饲料系数等生长性能指标虽无显著影响(P>0.05),但与对照组相比,添加1.5%的复方中草药制剂C可使吉富罗非鱼的增重率提高10.82%,饲料系数降低4.71%,蛋白质效率提高15.68%;三种复方中草药制剂均可显著促进罗非鱼肠道细菌、双歧杆菌、乳酸杆菌的生长(P<0.05),C方中草药制剂还可显著抑制大肠杆菌生长(P<0.05)。结果表明,C方中草药制剂可显著促进罗非鱼肠道中乳酸杆菌等有益菌群的增殖,抑制大肠杆菌生长,从而有效促进罗非鱼生长。     

Effects of Dietary Stachyose on Growth Performance,Digestive Enzyme Activities and Intestinal Morphology of Juvenile Turbot(Scophthalmus maximus L)

A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot(Scophthalmus maximus L). Five isonitrogenous(49.58% crude protein) and isolipidic(10.50% crude lipid) diets were formulated to contain 0(Control), 0.625%(S-0.625), 1.25%(S-1.25), 2.5%(S-2.5) and 5%(S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly(P 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher(P 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose(P 0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased(P 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose(1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.     

Study on biogenic amines in various dry salted fish consumed in China

This study was carried out to investigate the biogenic amines（BAs）, physicochemical property and microorganisms in dry salted fish, a traditional aquatic food consumed in China. Forty three samples of dry salted fish were gathered from retail and wholesale markets and manufacturers, which had been produced in various regions in China. Cadaverine（CAD） and putrescine（PUT） were quantitatively the most common biogenic amines. About 14% of the samples exceeded the histamine content standards established by the FDA and/or EU. The highest histamine content was found in Silver pomfret（Pampus argenteus）(347.79 mg kg^-1). Five of forty three samples exceeded the acceptable content of TYR(100 mg kg^-1), and 23.26% of dried-salted fish contained high contents of biogenic amines(above 600 mg kg^-1). In addition, species, regions, pickling processes and drying methods made the physicochemical property, microorganisms and biogenic amines in dry salted fish to be different to some extents. The total plate count（TPC） was much higher than that of total halophilic bacteria in all samples. The biogenic amines, physicochemical property and microbiological counts exhibited large variations among samples. Furthermore, no significant correlation between biogenic amines and physicochemical property and TPC was observed. This study indicated that dry salted fish may still present healthy risk for BAs, depending on the processing methods, storage conditions among others.     

Effect of hypoxia on the blood of large yellow croaker (Pseudosciaena crocea)

Large areas of hypoxic water have recently been reported in the East China Sea. It is hypothesized that hypoxia may be partially responsible for the decline of some fish stocks. We evaluated the effect of hypoxia on large yellow croaker (Pseudosciaena crocea). The fish were exposed to three concentrations of dissolved oxygen (DO; 1.5 mg/L and 2.0 mg/L, and 6.5 mg/L control). We collected blood after 6, 12, 24, 48, and 96 h exposure. There was a significant increase in red blood count, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin in the group exposed to 1.5 mg/L DO after 6 h or 12 h, and a delayed increase (only elevated after 48 h and 96 h) in these indices in the group exposed to 2.0 mg/L DO. Plasma glucose concentrations increased significantly in both hypoxic groups after 24 h. Furthermore, plasma lactate and lactate dehydrogenase activity increased significantly after the first 6 h exposure in both hypoxic groups. Our results suggest that large yellow croakers could not maintain the aerobic pathway and instead use anaerobic metabolism for survival when DO levels fall below 2.0 mg/L. We conclude that the occurrence of hypoxia (<2 mg/L DO) in the East China Sea could cause metabolic stress for large yellow croakers and may be partially responsible for the population decline of this species.     

Isolation of a novel strain of Cyanobacterium sp. with good adaptation to extreme alkalinity and high polysaccharide yield

The use of high alkaline medium is a feasible way to provide carbon source and prevent biological contamination for the outdoor cultivation of alkaliphilic microalgae and cyanobacteria. A novel cyanobacterial strain was isolated from the open pond of a marine green alga( Picochlorum sp. SCSIO-45015, Sanya, Hainan) and identified as Cyanobacterium sp. SCSIO-45682. The effects of initial sodium bicarbonate(NaHCO_3) concentrations on the growth and biochemical composition of Cyanobacterium sp. SCSIO-45682 were investigated. The results demonstrated that Cyanobacterium sp. SCSIO-45682 had good adaptation to 16.8-g/L NaHCO_3(the same concentration of NaHCO_3 used in Zarrouk medium for Spirulina). Moreover, the yields of biomass, polysaccharide, chlorophyll a(chl a), and phycocyanin increased under high NaHCO_3 concentrations. The maximum final biomass concentration of 2.5 g/L was observed at 8.4-g/L NaHCO_3, while the highest intracellular total saccharide content of 49.2% of dry weight(DW) and exopolysaccharide(EPS) concentration of 93 mg/L were achieved at the NaHCO_3 concentration of 16.8 g/L. The crude protein content declined under high NaHCO_3 concentrations, which provide a possible explanation for the accumulation of polysaccharide. This study shows a good potential of alkaliphilic Cyanobacterium sp. SCSIO-45682 as a polysaccharide feedstock.     

Fucoxanthin Isolated from Undaria pinnatifida Can Interact with Escherichia coli and lactobacilli in the Intestine and Inhibit the Growth of Pathogenic Bacteria

Fucoxanthin is a xanthophyll-type carotenoid that provides many benefits to human health. However, the mechanism by which fucoxanthin interacts with microbes and inhibits pathogenic bacteria is unknown. In this study, we investigated the effects of fucoxanthin isolated from the edible seaweed Undaria pinnatifida on pathogenic bacteria Escherichia coli and lactobacilli both in vitro and in vivo. Fucoxanthin strongly inhibited the growth of Gram-positive pathogenic bacteria but was less effective against Gram-negative bacteria. Fucoxanthin extracted from the crude mixture had a recovery rate of 93.38% and a purity of 82.70%, which were higher than those of fucoxanthin extracted using a previous method. Fucoxanthin also promoted the growth of intestinal microbes in mice. Fucoxanthinol, a metabolite of fucoxanthin, was generated in the culture media. Fucoxanthin can be deacetylated into fucoxanthinol not only by conventional digestive enzymes in the digestive tract, but also by E. coli and lactobacilli in the intestine. These results indicate that fucoxanthin interacts with and influences E. coli and lactobacilli in the intestine. Therefore, fucoxanthin isolated from Undaria pinnatifida possibly can be applied in human health maintenance.