碳氮比调节在对虾养殖中的作用及优化
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摘要
目前随着我国对虾养殖产业现代化、工场化和集约化程度的发展,高密度的养殖模式使得养殖环境生态系统结构单一,抵抗能力薄弱,长期使用的药物也改变了对虾肠道菌群的正常组成,导致了肠道内微生态环境的失调,同时使药物在养殖生物体内形成残留。近年来应用范围越来越广泛的碳氮比调节(C/N)和有益菌添加等技术已在对虾养殖业中发挥着越来越重要的作用,不仅从根本上完善和优化了养殖微生态系统,改善了水质状况,还可以降低饵料成本,提高对虾养殖产量。但是C/N调节技术对养殖环境中特定细菌种群和浮游动物的影响还有待研究,同时有益菌添加技术也面临着有益菌不能长时间维持,很难形成优势菌种等诸多问题。因此本文主要针对以上问题设计了如下实验内容。
本文主要研究了以下几个方面内容：1.C/N调节在凡纳滨对虾(Litopenaeus vannamei)养殖中的作用,具体包括对水质因子、对虾产量、浮游动物、异养细菌和氮循环菌的影响；2.对虾养殖环境中多种不同种类的细菌对环境C/N的响应,以及有益菌对有害菌的抑制作用,3.如何利用产纤维素酶细菌对植物纤维素的分解作用来优化C/N调节技术,降低技术成本。作者期望通过本实验将C/N调节与有益菌添加两种手段有效的结合在一起,通过向养殖水体中添加碳源来促进养殖环境中有益菌的生长,从而降低有毒无机氮化合物的水平,抑制有害细菌的繁殖,并可以形成大量生物絮团,为对虾的生长提供良好的环境。本实验的主要研究结果如下：
1.碳源添加在对虾养殖中的作用
本实验研究了在凡纳滨对虾零换水养殖系统中通过添加碳源对水质指标、对虾生长、浮游动物密度、环境C/N、水体和底质中的总菌和氮循环菌密度等的影响。将蔗糖作为碳源向养殖环境中进行添加,共分5个水平：理论碳源添加量((?)CH=0.465×feed×(42%/30%))的0%、25%、50%、75%和100%。我们的实验结果表明通过添加碳源,特别是75%和100%的理论碳源添加量可以在养殖后期有效的抑制氨氮(TAN)和亚硝氮(N02-N)在水体中的积累(P<0.05)。碳源添加对养殖水体中硝氮(N03-N)、叶绿素a、COD和BOD的浓度水平没有显著的影响。此外碳源添加可以明显提高水体中的C/N和总菌的密度水平(P<0.05),但对于底质中的C/N和总菌密度水平的影响不明显。我们还发现通过添加碳源可以明显影响水体和底质中氮循环菌(亚硝酸细菌、硝酸细菌和反硝化细菌)的密度。碳源添加还可以明显促进浮游动物的生长(P<0.05)。根据本次实验结果我们可以得出结论：75%的理论碳源添加量最适于对虾的生长,包括提高产量、成活率和降低饵料系数。因此,向凡纳滨对虾零换水养殖系统中添加碳源可以有效的改善水质,促进细菌和浮游动物生长,进而为对虾生长提供有利的条件。
2.对虾养殖环境中细菌在C/N限制条件下的筛选和鉴定
本实验从我国北部沿海地下水养殖环境,中部沿海和南部沿海的多种凡纳滨对虾养殖环境的水体和底质中富集分离出多种细菌,并利用化学方法进行了初步鉴定,共筛选出芽孢杆菌37株,乳酸菌42株,弧菌50株,亚硝酸细菌50株,硝酸细菌60株,反硝化细菌10株。我们进一步利用碳限制和氮限制两种环境进行细菌培养,筛选出对限定C/N条件响应较明显的菌株,并通过16S rDNA序列分析技术对其进行了分析和鉴定,最终共得到每种细菌各4株,鉴定芽孢杆菌、乳酸菌、弧菌、亚硝酸细菌、硝酸细菌和反硝化细菌分别属于芽孢杆菌属、肠球菌属、弧菌属、亚硝化单胞菌属、硝化杆菌属和交替假单胞菌属。本实验所筛选的多种菌株为下一步研究其对培养环境C/N的响应和今后进一步的工作奠定了基础。
3.水体C/N对细菌生长及拮抗作用的影响
本实验将从凡纳滨对虾养殖池塘中采集分离出的多株芽孢杆菌、乳酸菌、弧菌、亚硝酸细菌、硝酸细菌和反硝化细菌在不同水体C/N(2、5、10、15、20)的环境下进行培养,研究养殖环境营养水平下六种细菌的生长,以及芽孢杆菌、乳酸菌和弧菌的竞争水平及细菌菌体C/N的变化。研究发现,芽孢杆菌在水体C/N=15时生长水平最好(密度最高为6.7×107cells/ml),乳酸菌适宜在较高的水体C/N下生长(水体C/N=10、15、20；密度最高为2.6×107cells/ml),弧菌适宜在较低的水体C/N下生长(水体C/N=5、10；密度最高为5.1×107cells/ml),亚硝酸细菌和硝酸细菌对C/N的响应不明显,反硝化细菌在水体C/N=10的环境中生长较好(密度最高为2.6×107cells/ml).在芽孢杆菌与弧菌混合培养的实验中,当水体C/N=10、15与20时,培养24h时芽孢杆菌密度>2.5×107cells/ml,明显高于弧菌密度(P<0.05),且在培养基环境和模拟养殖环境中实验结果相似。在乳酸菌对弧菌的抑制实验中,当水体C/N=10与15时乳酸菌培养上清液对弧菌生长的抑制作用达到较高水平,抑菌圈直径分别为3.2与3.1cm,明显高于其它处理组(P<0.05)。芽孢杆菌、乳酸菌与弧菌的菌体C/N均与水体C/N的变化成正相关,最低分别为5.41、3.92与5.44；最高分别为7.27、5.07与15.35。以上结果表明,较高的水体C/N适合芽孢杆菌(水体C/N=15)和乳酸菌(水体C/N=10)的生长,较低的水体C/N适合弧菌(水体C/N=5或10)的生长,并且当水体C/N较高时芽孢杆菌和乳酸菌可以明显抑制弧菌的生长,并且在模拟养殖环境中芽孢杆菌对弧菌的抑制作用与培养基环境相似。因此向养殖水体中添加碳源,将水体的C/N提高至超过10的水平不仅可以促进芽孢杆菌、乳酸菌和反硝化细菌的快速生长,同时还可以起到有效抑制弧菌繁殖的作用。
4.产纤维素酶细菌的筛选及其分解碳源能力的研究
本实验从凡纳滨对虾的多种养殖环境(高密度高位池养殖池塘、添加甘蔗渣的高密度温棚养殖池塘、粗放式养殖池塘)、生态净水池以及排污池中富集分离出多株产纤维素酶细菌,并筛选出一株具有较全酶系和对纤维素具有较高分解能力的菌株GS-2。所筛选产纤维素酶细菌的刚果红染色透明圈直径及其与菌落直径的比值范围分别为12.3-41.7mm和2.8-15.6,FPA、CMCase和B-葡萄糖苷酶的酶活力范围分别为2.30-8.03、2.34-7.43和2.03-34.98U/ml；菌株GS-2的刚果红染色透明圈直径及其与菌落直径的比值分别为41.7mm和15.6,其FPA、CMCase和B-葡萄糖苷酶的酶活力分别为8.03,7.43和3.92U/ml,通过16S rDNA序列同源性比较确定其为短小芽孢杆菌(Bacillus pumilus)。菌株GS-2产酶最适的发酵液起始pH为8,最适温度为30℃,最适培养时间为30h,且适合在有氧条件下进行产酶代谢,在厌氧条件下产酶效率较低。菌株GS-2对微晶纤维素(Avicel)的分解效率最高,在培养基环境下可达到21.24%,其次为稻草和稻壳,同时该菌的分解Avicel的产物可以显著提高环境的溶解态碳含量和水体C/N(217.33mg/1；59.27),并且能够明显促进芽孢杆菌菌株Z5的生长。因此通过向养殖环境中投放产纤维素酶细菌与适合的天然纤维素底物,不仅可以实现其对底物的不断分解,为养殖环境提供碳源,提高水体C/N,还可以促进芽孢杆菌的生长,改善养殖微生态环境。
This experiment was conduct to study the issues as follows:1. The effect of adjusting C/N ratio on the culture of Litopenaeus vannamei, i.e. heterotrophic bacteria, nitrogen cycle bacteria, zooplankton, water quality and shrimp growth performance;2. The response of the bacterial growth to the C/N ratio and the inhibition of probiotic bacteria on the harmful bacteria.3. How to improve the technology of adjusting C/N ratio according to the cellulose decomposing of cellulose-degrading bacterium. The primary results were listed below.
1. Effects of carbohydrate addition on shrimp culture system
The present study investigated the effects of carbohydrate addition on the water quality, carbon/nitrogen ratios, densities of total bacteria, nitrogen cycle bacteria and zooplankton, and shrimp growth performance in a zero-water exchange system for Litopenaeus vannamei intensive culture. Sucrose was added as the carbohydrate into the water at five levels, i.e.,0,25%,50%,75%and100%of the theoretical adding quantity. Our results demonstrated that carbohydrate addition, especially75%and100%of the theoretical quantity significantly reduced the concentrations of total ammonia nitrogen and nitrite nitrogen during the late culture period in the water (P<0.05). Carbohydrate addition did not significantly affect the concentration of nitrate nitrogen, chlorophyll-a, COD and BOD. In addition, carbohydrate addition significantly increased carbon/nitrogen ratios and total bacterial densities of the water (P<0.05), whereas did not affect those of the sediment on the whole. We also found that the densities of nitrogen cycle bacteria (ammonia-oxidizing, nitrite-oxidizing and denitrifying bacteria) in both the water and the sediment of the carbohydrate-added treatments were obviously affected compared to those of the control treatments. Zooplankton densities in the water were determined to be significantly increased by carbohydrate addition (P<0.05). The parameters for shrimp growth performance including the yield, survival and feed conversion ratio demonstrated that75%of the theoretical quantity was more suitable for L. vannamei culture. Conclusively, carbohydrate addition into zero-water exchange systems for L. vannamei intensive culture can effectively improve the water quality, bacterial activities and zooplankton growth, consequently resulting in the better growth performance.
2. Isolation and identification of several bacteria in the culture system of Litopenaeus vannamet
In this study, we isolated several bacteria from different shrimp culture system, i.e. the north coast ground-water culture system, the mid coast seawater culture system and the south coast seawater culture system, respectively. We identified these bacteria using chemical method preliminarily, and further isolated the strain which has more significant response to the C/N ratio. Six types of bacteria were isolated, i.e. Bacillus, lactic acid bacteria, Vibrio, ammonia-oxidizing, nitrite-oxidizing and denitrifying bacteria, respectively. According to the16S rDNA sequence analysis, they were indentified as Bacillus sp., Enterococcus sp., Vibrio sp., Nitrosomonas sp., Nitrobacter sp. and Pseudoalteromonas sp., respectively. The bacteria isolated in this part were prepared for the study of the response of bacteria growth to the C/N ratio and the futher work.
3. The effect of C:N ratio of the water on the growth and competition of bacteria
From the Litopenaeus vannamei culture ponds, the12strains of Bacillus, lactic acid bacteria and Vibrio were isolated which performed better growth in the conditions of carbon-limited (C:N=2) or nitrogen-limited (C:N=20). The levels of C:N ratio of the water (C:NW=2,5,10,15,20) were set to determine its effects on the growth, competition and the cell C:N ratio of the bacteria. It was found that the appropriate C:NW for the growth of Bacillus, lactic acid bacteria, Vibrio and denitrifying bacteria were C:Nw=15, C:Nw=10, C:Nw=5or10and C:Nw=10, respectively; and the highest values were determined as6.7×107,2.6×107,5.1×107and2.6×107cells/ml, respectively. The C:NW did not significantly effect the growth of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. After24h culturing of Bacillus and Vibrio together in the treatments with C:Nw=10,15and20, the densities of Bacillus reached2.5×10'cells/ml, significantly higher than those of Vibrio (<2.0×107cells/ml;<0.05), and the experiment under the imitated culture environment had the same result with the media environment. The inhibition of lactic acid bacteria extracellular products on the growth of Vibrio were significantly higher in the treatments with C:Nw=10and15than that in the other treatments (P<0.05). The C:N ratios of the body composition of Bacillus, lactic acid bacteria and Vibrio were increased with the C:NW, in range of5.41-7.27,3.92-5.07and5.44-15.35, respectively. Conclusively, adjusting the C:N ratio in the water above10with carbohydrate addition not only improved the growth of Bacillus and lactic acid bacteria, but also inhibited the Vibrio growth.
4. Isolation and carbohydrate-hydrolyzing-capacity study of cellulose-degrading bacterium
In this study, through isolation from the Litopenaeus vannamei culture environment and selective enrichment culture, a cellulose-degrading bacterium, strain GS-2was isolated. Clear zones which showed cellulose hydrolysis were41.7mm in diameter, and the ratio to bacteria growth diameter was15.6. The enzyme activity of exoglucanase, endoglucanase and B-Glucosidases were8.03,7.43and3.92U/ml, respectively. According to the16S rDNA sequence analysis, it was indentified as Bacillus pumilus. The optimum pH of the initial culture substrate for the enzyme production was8, an optimum temperature30℃and an optimum culture time30h. The decomposed efficiency of strain GS-2was highest with the substrate of Avicel, which was21.24%in the culture medium. Futhermore, the production of decomposing Avicel could significantly increase the dissolved carbon concentration and C/N ratio (217.33mg/1;59.27) of the environment, and improve the growth of Bacillus. Conclusively, adding the cellulose-degrading bacterium and nature cellulose into the culture system can keep the cellulose decomposing continuous, provide more carbon source and increase the C/N ratio, and improve the growth of Bacillus and the water quality.
本文主要研究了以下几个方面内容：1.C/N调节在凡纳滨对虾(Litopenaeus vannamei)养殖中的作用,具体包括对水质因子、对虾产量、浮游动物、异养细菌和氮循环菌的影响；2.对虾养殖环境中多种不同种类的细菌对环境C/N的响应,以及有益菌对有害菌的抑制作用,3.如何利用产纤维素酶细菌对植物纤维素的分解作用来优化C/N调节技术,降低技术成本。作者期望通过本实验将C/N调节与有益菌添加两种手段有效的结合在一起,通过向养殖水体中添加碳源来促进养殖环境中有益菌的生长,从而降低有毒无机氮化合物的水平,抑制有害细菌的繁殖,并可以形成大量生物絮团,为对虾的生长提供良好的环境。本实验的主要研究结果如下：
1.碳源添加在对虾养殖中的作用
本实验研究了在凡纳滨对虾零换水养殖系统中通过添加碳源对水质指标、对虾生长、浮游动物密度、环境C/N、水体和底质中的总菌和氮循环菌密度等的影响。将蔗糖作为碳源向养殖环境中进行添加,共分5个水平：理论碳源添加量((?)CH=0.465×feed×(42%/30%))的0%、25%、50%、75%和100%。我们的实验结果表明通过添加碳源,特别是75%和100%的理论碳源添加量可以在养殖后期有效的抑制氨氮(TAN)和亚硝氮(N02-N)在水体中的积累(P<0.05)。碳源添加对养殖水体中硝氮(N03-N)、叶绿素a、COD和BOD的浓度水平没有显著的影响。此外碳源添加可以明显提高水体中的C/N和总菌的密度水平(P<0.05),但对于底质中的C/N和总菌密度水平的影响不明显。我们还发现通过添加碳源可以明显影响水体和底质中氮循环菌(亚硝酸细菌、硝酸细菌和反硝化细菌)的密度。碳源添加还可以明显促进浮游动物的生长(P<0.05)。根据本次实验结果我们可以得出结论：75%的理论碳源添加量最适于对虾的生长,包括提高产量、成活率和降低饵料系数。因此,向凡纳滨对虾零换水养殖系统中添加碳源可以有效的改善水质,促进细菌和浮游动物生长,进而为对虾生长提供有利的条件。
2.对虾养殖环境中细菌在C/N限制条件下的筛选和鉴定
本实验从我国北部沿海地下水养殖环境,中部沿海和南部沿海的多种凡纳滨对虾养殖环境的水体和底质中富集分离出多种细菌,并利用化学方法进行了初步鉴定,共筛选出芽孢杆菌37株,乳酸菌42株,弧菌50株,亚硝酸细菌50株,硝酸细菌60株,反硝化细菌10株。我们进一步利用碳限制和氮限制两种环境进行细菌培养,筛选出对限定C/N条件响应较明显的菌株,并通过16S rDNA序列分析技术对其进行了分析和鉴定,最终共得到每种细菌各4株,鉴定芽孢杆菌、乳酸菌、弧菌、亚硝酸细菌、硝酸细菌和反硝化细菌分别属于芽孢杆菌属、肠球菌属、弧菌属、亚硝化单胞菌属、硝化杆菌属和交替假单胞菌属。本实验所筛选的多种菌株为下一步研究其对培养环境C/N的响应和今后进一步的工作奠定了基础。
3.水体C/N对细菌生长及拮抗作用的影响
本实验将从凡纳滨对虾养殖池塘中采集分离出的多株芽孢杆菌、乳酸菌、弧菌、亚硝酸细菌、硝酸细菌和反硝化细菌在不同水体C/N(2、5、10、15、20)的环境下进行培养,研究养殖环境营养水平下六种细菌的生长,以及芽孢杆菌、乳酸菌和弧菌的竞争水平及细菌菌体C/N的变化。研究发现,芽孢杆菌在水体C/N=15时生长水平最好(密度最高为6.7×107cells/ml),乳酸菌适宜在较高的水体C/N下生长(水体C/N=10、15、20；密度最高为2.6×107cells/ml),弧菌适宜在较低的水体C/N下生长(水体C/N=5、10；密度最高为5.1×107cells/ml),亚硝酸细菌和硝酸细菌对C/N的响应不明显,反硝化细菌在水体C/N=10的环境中生长较好(密度最高为2.6×107cells/ml).在芽孢杆菌与弧菌混合培养的实验中,当水体C/N=10、15与20时,培养24h时芽孢杆菌密度>2.5×107cells/ml,明显高于弧菌密度(P<0.05),且在培养基环境和模拟养殖环境中实验结果相似。在乳酸菌对弧菌的抑制实验中,当水体C/N=10与15时乳酸菌培养上清液对弧菌生长的抑制作用达到较高水平,抑菌圈直径分别为3.2与3.1cm,明显高于其它处理组(P<0.05)。芽孢杆菌、乳酸菌与弧菌的菌体C/N均与水体C/N的变化成正相关,最低分别为5.41、3.92与5.44；最高分别为7.27、5.07与15.35。以上结果表明,较高的水体C/N适合芽孢杆菌(水体C/N=15)和乳酸菌(水体C/N=10)的生长,较低的水体C/N适合弧菌(水体C/N=5或10)的生长,并且当水体C/N较高时芽孢杆菌和乳酸菌可以明显抑制弧菌的生长,并且在模拟养殖环境中芽孢杆菌对弧菌的抑制作用与培养基环境相似。因此向养殖水体中添加碳源,将水体的C/N提高至超过10的水平不仅可以促进芽孢杆菌、乳酸菌和反硝化细菌的快速生长,同时还可以起到有效抑制弧菌繁殖的作用。
4.产纤维素酶细菌的筛选及其分解碳源能力的研究
本实验从凡纳滨对虾的多种养殖环境(高密度高位池养殖池塘、添加甘蔗渣的高密度温棚养殖池塘、粗放式养殖池塘)、生态净水池以及排污池中富集分离出多株产纤维素酶细菌,并筛选出一株具有较全酶系和对纤维素具有较高分解能力的菌株GS-2。所筛选产纤维素酶细菌的刚果红染色透明圈直径及其与菌落直径的比值范围分别为12.3-41.7mm和2.8-15.6,FPA、CMCase和B-葡萄糖苷酶的酶活力范围分别为2.30-8.03、2.34-7.43和2.03-34.98U/ml；菌株GS-2的刚果红染色透明圈直径及其与菌落直径的比值分别为41.7mm和15.6,其FPA、CMCase和B-葡萄糖苷酶的酶活力分别为8.03,7.43和3.92U/ml,通过16S rDNA序列同源性比较确定其为短小芽孢杆菌(Bacillus pumilus)。菌株GS-2产酶最适的发酵液起始pH为8,最适温度为30℃,最适培养时间为30h,且适合在有氧条件下进行产酶代谢,在厌氧条件下产酶效率较低。菌株GS-2对微晶纤维素(Avicel)的分解效率最高,在培养基环境下可达到21.24%,其次为稻草和稻壳,同时该菌的分解Avicel的产物可以显著提高环境的溶解态碳含量和水体C/N(217.33mg/1；59.27),并且能够明显促进芽孢杆菌菌株Z5的生长。因此通过向养殖环境中投放产纤维素酶细菌与适合的天然纤维素底物,不仅可以实现其对底物的不断分解,为养殖环境提供碳源,提高水体C/N,还可以促进芽孢杆菌的生长,改善养殖微生态环境。
This experiment was conduct to study the issues as follows:1. The effect of adjusting C/N ratio on the culture of Litopenaeus vannamei, i.e. heterotrophic bacteria, nitrogen cycle bacteria, zooplankton, water quality and shrimp growth performance;2. The response of the bacterial growth to the C/N ratio and the inhibition of probiotic bacteria on the harmful bacteria.3. How to improve the technology of adjusting C/N ratio according to the cellulose decomposing of cellulose-degrading bacterium. The primary results were listed below.
1. Effects of carbohydrate addition on shrimp culture system
The present study investigated the effects of carbohydrate addition on the water quality, carbon/nitrogen ratios, densities of total bacteria, nitrogen cycle bacteria and zooplankton, and shrimp growth performance in a zero-water exchange system for Litopenaeus vannamei intensive culture. Sucrose was added as the carbohydrate into the water at five levels, i.e.,0,25%,50%,75%and100%of the theoretical adding quantity. Our results demonstrated that carbohydrate addition, especially75%and100%of the theoretical quantity significantly reduced the concentrations of total ammonia nitrogen and nitrite nitrogen during the late culture period in the water (P<0.05). Carbohydrate addition did not significantly affect the concentration of nitrate nitrogen, chlorophyll-a, COD and BOD. In addition, carbohydrate addition significantly increased carbon/nitrogen ratios and total bacterial densities of the water (P<0.05), whereas did not affect those of the sediment on the whole. We also found that the densities of nitrogen cycle bacteria (ammonia-oxidizing, nitrite-oxidizing and denitrifying bacteria) in both the water and the sediment of the carbohydrate-added treatments were obviously affected compared to those of the control treatments. Zooplankton densities in the water were determined to be significantly increased by carbohydrate addition (P<0.05). The parameters for shrimp growth performance including the yield, survival and feed conversion ratio demonstrated that75%of the theoretical quantity was more suitable for L. vannamei culture. Conclusively, carbohydrate addition into zero-water exchange systems for L. vannamei intensive culture can effectively improve the water quality, bacterial activities and zooplankton growth, consequently resulting in the better growth performance.
2. Isolation and identification of several bacteria in the culture system of Litopenaeus vannamet
In this study, we isolated several bacteria from different shrimp culture system, i.e. the north coast ground-water culture system, the mid coast seawater culture system and the south coast seawater culture system, respectively. We identified these bacteria using chemical method preliminarily, and further isolated the strain which has more significant response to the C/N ratio. Six types of bacteria were isolated, i.e. Bacillus, lactic acid bacteria, Vibrio, ammonia-oxidizing, nitrite-oxidizing and denitrifying bacteria, respectively. According to the16S rDNA sequence analysis, they were indentified as Bacillus sp., Enterococcus sp., Vibrio sp., Nitrosomonas sp., Nitrobacter sp. and Pseudoalteromonas sp., respectively. The bacteria isolated in this part were prepared for the study of the response of bacteria growth to the C/N ratio and the futher work.
3. The effect of C:N ratio of the water on the growth and competition of bacteria
From the Litopenaeus vannamei culture ponds, the12strains of Bacillus, lactic acid bacteria and Vibrio were isolated which performed better growth in the conditions of carbon-limited (C:N=2) or nitrogen-limited (C:N=20). The levels of C:N ratio of the water (C:NW=2,5,10,15,20) were set to determine its effects on the growth, competition and the cell C:N ratio of the bacteria. It was found that the appropriate C:NW for the growth of Bacillus, lactic acid bacteria, Vibrio and denitrifying bacteria were C:Nw=15, C:Nw=10, C:Nw=5or10and C:Nw=10, respectively; and the highest values were determined as6.7×107,2.6×107,5.1×107and2.6×107cells/ml, respectively. The C:NW did not significantly effect the growth of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. After24h culturing of Bacillus and Vibrio together in the treatments with C:Nw=10,15and20, the densities of Bacillus reached2.5×10'cells/ml, significantly higher than those of Vibrio (<2.0×107cells/ml;<0.05), and the experiment under the imitated culture environment had the same result with the media environment. The inhibition of lactic acid bacteria extracellular products on the growth of Vibrio were significantly higher in the treatments with C:Nw=10and15than that in the other treatments (P<0.05). The C:N ratios of the body composition of Bacillus, lactic acid bacteria and Vibrio were increased with the C:NW, in range of5.41-7.27,3.92-5.07and5.44-15.35, respectively. Conclusively, adjusting the C:N ratio in the water above10with carbohydrate addition not only improved the growth of Bacillus and lactic acid bacteria, but also inhibited the Vibrio growth.
4. Isolation and carbohydrate-hydrolyzing-capacity study of cellulose-degrading bacterium
In this study, through isolation from the Litopenaeus vannamei culture environment and selective enrichment culture, a cellulose-degrading bacterium, strain GS-2was isolated. Clear zones which showed cellulose hydrolysis were41.7mm in diameter, and the ratio to bacteria growth diameter was15.6. The enzyme activity of exoglucanase, endoglucanase and B-Glucosidases were8.03,7.43and3.92U/ml, respectively. According to the16S rDNA sequence analysis, it was indentified as Bacillus pumilus. The optimum pH of the initial culture substrate for the enzyme production was8, an optimum temperature30℃and an optimum culture time30h. The decomposed efficiency of strain GS-2was highest with the substrate of Avicel, which was21.24%in the culture medium. Futhermore, the production of decomposing Avicel could significantly increase the dissolved carbon concentration and C/N ratio (217.33mg/1;59.27) of the environment, and improve the growth of Bacillus. Conclusively, adding the cellulose-degrading bacterium and nature cellulose into the culture system can keep the cellulose decomposing continuous, provide more carbon source and increase the C/N ratio, and improve the growth of Bacillus and the water quality.
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