【作者】 赵培；

【导师】 黄倢；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 博士

【摘要】 自上世纪70年代,水产养殖业作为增加蛋白质来源最迅速最可靠的方式发展迅速,水产品产量平均每年的增长速率达8.9-9.1%,有效解决了发展中国家的蛋白来源匮乏问题。据世界粮农组织报道,过去50年水产养殖产量已增长近40倍,未来50年至少增长5倍才能满足人类日益增长的需要。然而,随着养殖规模的扩大和养殖密度的提高,环境污染和经济制约等问题日益严重,极大限制了水产养殖业的发展。特别是在集约化水产养殖过程中,大量残饵粪便排放到养殖水体内,使氨氮等有毒物质迅速累积,导致养殖水质恶化,并产生潜在的环境污染。很长一段时间内,解决水质恶化的最有效的方法是及时进行排水换水,然而,仅仅是小型或者中型的水产养殖系统每天的用水量就达几百方,南美白对虾每生长1kg,至少需要20 m3的换水量,一个中型的跑道式养殖系统(140m3)的日换水总量大约是养殖池水总量的100倍。此外,还可通过基于不同生物处理模式的循环水养殖系统来消除养殖水体污染物,日换水量降至养殖水体总量的10%,极大节约了水资源,然而该系统前期建设成本过高,资本金回收时间长,同时该系统运行过程中需要消耗大量的人力物力资源,加上前期的建设成本,该系统的养殖成本相对于传统的养殖成本不但没有降低,反而提高了近1/3。如何改变这个资源消耗线性发展的现状,一个很有发展前途的养殖技术就是所谓的生物絮团技术。生物絮团养殖系统中,养殖池内的异养菌和藻类在可控条件可形成絮团,该系统是将传统的水处理系统应用到水产养殖中,微生物吸收转化鱼体排泄物可有效去除养殖水体污染物,主要取决于异养菌的大量繁殖。异养菌消耗1g有机碳可生产0.4g菌体蛋白,取决于细菌转化有机氮所需的C/N比,微生物本身C/N比为4,碳水化合物的含碳量为50%,因此转化1g有机氮至少需要消耗20g碳水化合物。传统的水产养殖系统内微生物对残余饵料及有机废物的转化率是极低的,仅有7%的氮和6%的磷被微生物转化,但是如果养殖水体的碳和氮被很好的调控,微生物对残饵粪便的转化率就会明显提高。生物絮团技术在国外已应用到罗非鱼、凡纳滨对虾、罗氏沼虾和斑节对虾等的养殖,但日本囊对虾的养殖中该技术的应用还未见报道。本论文通过添加蔗糖将生物絮团技术应用到日本囊对虾高密度精养系统中,研究整个养殖周期内生物絮团技术对日本囊对虾生长、存活及水质调控的影响,进一步分析生物絮团内微生物的种群多样性,确定生物絮团的优势菌群。结果发现通过添加蔗糖,养殖水体氨氮和亚硝酸盐浓度得以显著降低,日本囊对虾的饵料转化率和蛋白利用率高达1.67和1.42,对虾生长速度显著提高,对虾存活率高达65.7%,最终实现了1.3kg m-2的养殖产量。PCR-DGGE分析生物絮团内微生物的种群多样性,发现生物絮团组的优势菌群为Probacterium和Bacillus sp.,而传统对照组的优势菌群为Probacterium和Vibrio sp.,因此通过添加蔗糖形成的生物絮团内含益生菌Bacillus sp.,在一定程度上抑制了养殖水体内致病菌Vibrio sp.的繁殖。然而通过添加蔗糖培养生物絮团所需养殖成本过高,不适于大规模推广。本论文尝试利用农业副产品(秸秆、麦麸、豆粕、花生粕、玉米粉等)发酵生产坚强芽孢杆菌,通过独立配方试验筛选出最佳发酵培养基成分,采用单因素和正交试验对培养基和发酵条件进行优化,建立简易可行的有益微生物发酵工艺,并将发酵菌液应用到生物絮团强化培养过程中,为生物絮团培养提供有效的可溶性碳源,PCR-DGGE分析生物絮团内微生物的种群多样性及动态变化,确定发酵菌液添加所形成的益生菌优势,结果表明:采用小麦秸秆1.8%、麦麸4%、花生粕0.6%的配方,在初始pH5.4—7.2、温度30—40℃、装液率50%的条件下发酵37—102h得到的菌体数量最高。将发酵菌液按照100ppm d~ (-1)添加到生物絮团强化培养系统中,培养第3天即可形成生物絮团,而且絮团内微生物种群多样性显著升高,形成以芽孢杆菌为优势的微生物种群。在此基础上,将发酵菌液应用到不同海水养殖动物(日本囊对虾、凡纳滨对虾、中国对虾、刺参)的生物絮团养殖系统中,研究不同发酵菌液添加量对不同海水养殖动物生长、存活及水质调控的影响,分析生物絮团应用于不同养殖动物的最适发酵菌液添加量,建立不同养殖动物生物絮团的最适养殖工艺。结果表明,日本囊对虾生物絮团的最适养殖工艺:在养殖密度为200尾m-2的日本囊对虾养殖系统中,每天按照100ppm添加发酵菌液,整个养殖周期不换水。该系统中,生物絮团沉积量在养殖第11d即达0.3ml L~ (-1),养殖水体的氨氮和亚硝酸氮浓度均维持在0.1mg L~ (-1)以下,对虾生长速度达0.01g d~ (-1),养殖40d对虾成活率达65%。凡纳滨对虾生物絮团的最适养殖工艺:在养殖密度为300尾m-2的凡纳滨对虾养殖系统中,每天按照100ppm添加发酵菌液,整个养殖周期不换水。该系统中,生物絮团沉积量在养殖第11d即达0.5 ml L~ (-1),养殖水体的氨氮和亚硝酸氮浓度均维持在0.1 mg L~ (-1)以下,对虾生长速度达0.02g d~ (-1),养殖40d对虾成活率达87.5%。中国对虾生物絮团的最适养殖工艺:在养殖密度为100尾m-2的中国对虾养殖系统中,每天按照100ppm添加发酵菌液,整个养殖周期不换水。该系统中,生物絮团沉积量在第11d即达0.5 ml L~ (-1),养殖水体的氨氮和亚硝酸氮浓度均维持在0.1mg L~ (-1)以下,对虾生长速度达0.015g d~ (-1),养殖40d对虾成活率高达87.5%。刺参生物絮团的最适养殖工艺:在养殖密度为200头m-2的刺参养殖系统中,每天按照100ppm添加发酵菌液,整个养殖周期不换水。该系统中,生物絮团沉积量在养殖第11d即达0.55 ml L~ (-1),养殖水体的氨氮浓度维持在0.1mg/L以下,刺参生长速度最快,成活率最高为68.7%。发酵菌液应用于不同养殖动物的生物絮团养殖过程中,根据养殖水体的氨氮和亚硝酸氮浓度,养殖动物的生长速度以及成活率,采集具有显著功能性差异的生物絮团,进一步研究不同功能生物絮团的微生物种群差异,结果表明生物絮团中调控水质的原核生物可能包括脱硫菌Desulfobulbus japonicus、固氮菌Rhizobiales bacterium和亚硝酸氧化菌α-Proteobacterium;促进对虾生长的原核生物可能包括海洋光合细菌Roseobacter sp.和变形杆菌Proteobacterium;抗病的原核生物可能包括变性杆菌Proteobacterium、发光杆菌Photobacterium、拟杆菌Bacteroidetes bacterium和枯草芽孢杆菌Bacillus subtilis。总之,生物絮团技术可有效降低海水养殖系统的氨氮和亚硝酸水平,提高海水养殖动物的生长速度以及存活率,该作用效果可能与生物絮团内微生物的种群多样性有关。对特定的有益微生物进行简易发酵,并将其应用到生物絮团的强化培养系统中,可快速形成生物絮团并建立益生菌优势。通过添加发酵菌液将生物絮团技术应用到不同海水养殖动物系统中,研究不同海水养殖动物生物絮团养殖所需发酵菌液的最适添加量,建立不同海水养殖动物生物絮团的最适养殖工艺。采集具有显著功能性差异的生物絮团,研究不同功能生物絮团的微生物种群差异,分析不同功能生物絮团的优势微生物种群。本研究将为生物絮团养殖所需有益微生物的简易发酵提供特定的微生物资源,进而培养具有特定功能的生物絮团,为生物絮团技术在海水养殖中推广应用提供技术保障。更多还原

【Abstract】 The current worldwide growth rate of the aquaculture business (8.9–9.1% per year since the 1970s) is needed in order to copewith the problem of shortage in protein food supplies, which is particularly situated in the developing countries. According to Food Agriculture Organization, aquaculture production increased more than 40 times during the last 50 years and is expected to rise another 5 times in the coming 50 years. With the rapid expansion and intensi?cation, there is, however, also a growing concern about the ecological sustainability of shrimp culture. Environmental and economical limitations can hamper this growth. Especially intensive aquaculture coincides with the pollution of the culture water by an excess of organic materials and nutrients that are likely to cause acute toxic effects and long term environmental risks. For long, the most common method for dealing with this pollution has been the use of continuous replacement of the pond water with external fresh water. However, the water volume needed for even small to medium aquaculture systems can reach up to several hundreds of cubic meters per day. For instance, penaeid shrimp require about 20m3 fresh water per kg shrimp produced. For a medium-sized trout raceway systemof 140m3, even a daily replacement of 100 times the water volume is applied. A second approach is the removal of the major part of the pollutants in the water as is performed in recirculating aquaculture systems (RAS) with different kinds of biologically based water treatment systems. The amount of water that needs to be replaced on a daily basis generally is reduced to about 10% of the total water volume. However, this technique is costly in terms of capital investment. Operation of RAS furthermore increases energy and labour costs, so that taking all costs into consideration (investment plus operation costs) it can be estimated that unsustainable pond production can be performed at two thirds of the costs of RAS.A relatively new alternative to previous approaches is the bio-?ocs technology (BFT) aquaculture. In these systems, a co-culture of heterotrophic bacteria and algae is grown in ?ocs under controlled conditions within the culture pond. The system is based on the knowledge of conventional domestic wastewater treatment systems and is applied in aquaculture environments. Microbial biomass is grown on ?sh excreta resulting in a removal of these unwanted components from the water. The major driving force is the intensive growth of heterotrophic bacteria. They consume organic carbon; 1.0 g of carbohydrate-C yields about 0.4 g of bacterial cell dry weight-C; and depending on the bacterial C/N-ratio thereby immobilize mineral nitrogen. As such, Avnimelech calculated a carbohydrate need of 20 g to immobilize 1.0 g of N, based on a microbial C/N-ratio of 4 and a 50% C in dry carbohydrate. In integrated aquaculture systems using bacteria as additional nutrient trapping stage, the increase in retention by the use of bacteria is rather small. Schneider et al. stated that hardly 7% of the feed nitrogen and 6% of the feed phosphorus were retained by conversion in microbial biomass. However, when carbon and nitrogen are well balanced in the water solution and microbial assimilation of the ammonium is ef?ciently engineered, a complete retention can be obtained.This paper firstly used agricultural by-products (straw, wheat bran, soybean meal, peanut meal, corn powder) to ferment Bacillus firmus, and the fermentation standard of proteomics for bioflocs aquaculture was established: Add 1.8% straw, 4%wheat bran and 0.6% peanut meal together in a vessel, control the initial pH 5.4-7.2, temperature 30-40℃, liquid filling rate of 50% of 37~ (-1)02h fermentation, then fermentation fluid can achieve the best results.On this basis, the carbon source and fermentation liquid was added in the sea water aquaculture system, then the bioflocs formation, growth and survival of the aquaculture animals and water quality parameters were observed, finally the bioflocs technology of different seawater aquaculture animals were established. According to carbon addition, the optimal bioflocs technology of Litopenaeus vannamei was estabilished: Sugar was added to the aquculture system of Litopenaeus vannamei with C/N=20 at the density of 150 PL/m2 without water exchanging. In this system, bio-floc was formed on the 4th day, the content of ammonia nitrogen and nitrite was kept below 0.1 mg/L and 0.3mg/L, respectively, the growth rate was reached to 1mm/d, the survival rate was more than 80%. The production of L. vannamei was reach to(2.14±0.08)kg/m2 after 98-day cultivation by the bioflocs technology. According to fermentation fluid addition, the optimal bioflocs technology of Apostichopus japonicus was established: Fermentation fluid was added to the aquculture system of Apostichopus japonicus with 100ppm everyday without water exchanging. In this system, bio-floc was formed on the 11th day, the content of ammonia nitrogen a was kept below 0.1 mg/L, the growth rate was higher, and the survival rate was reached for 68.7%. According to carbon and fermentation fluid addition, the optimal bioflocs technology of Marsupenaeus japonicus was established: Sugar was added to the aquculture system of Marsupenaeus japonicu with C/N=60 at the density of 200 PL/m2 without water exchanging. In this system, bio-floc was formed on the 4th day, the content of ammonia nitrogen and nitrite was kept below 0.1 mg/L and 0.1mg/L, respectively, the growth rate was higher and the survival rate was more than 75%.Finally, the optimal bioflocs technology of Marsupenaeus japonicus was applied to the high-intensive, zero exchange aquaculture system of Marsupenaeus japonicus, the concentrations of ammonia-N and nitrite-N were both reduced, moreover, the feed conversion ratio and protein efficiency ratio were both increased up to 167% and 142%, shrimp growth was significantly increased and shrimp survival rate was increased to 65.7%, and ultimately the net yield was reach for 1.3kg/m2. By comparing the microbial diversity of bioflocs in traditional control and bioflocs treatment, the breeding of Vibrio sp. and Pseudoalteromonas sp. were probably inhabited by Bacillus sp. On the basis of this result, microbial diversity of different functional bioflocs was determined. The results were shown that the predominant microbe of bioflocs in the function of water control may include Desulfobulbus japonicus andα-Proteobacterium; the predominant microbe of bioflocs in the function of shrimp growth may include Roseobacter sp. and Proteobacterium; the predominant microbe of bioflocs in the function of disease control may include Proteobacterium, Photobacterium, Bacteroidetes bacterium and Bacillus subtilis.In conclusion, the application of bioflocs technology in high-intensive M. japonicus culture systems performed equally well as observed in other shrimp species. Bioflocs technology offers the possibility to simultaneously maintain a good water quality within aquaculture systems and produce additional food for shrimp. However, the optimal bioflocs technology of different aquaculture animals was different, probably due to the different living habits. In addition, the different function of bioflocs technology in seawater aquaculture system may be resulted from the microbial diversity of bioflocs.更多还原