The Role And Mechanisms Of Microbial Product And Exogenous Carbon In Affecting The Environment For Aquaculture

How to use microbial loop to improve the water quality in aquaculture is an increasing interest in recently years.Generally,the microbial manipulation in aquaculture includes addition of exogenous bacteria(microbial product)and addition of exogenous nutrient(carbon source).However,the efficacy of microbial products in improving aquaculture practices remains controversial,especially in the degradation of inorganic nitrogen.The lack of knowledge about the mechanism of how microbial product regulating the environment is to some extent an important reason for the above problem.The microbial product aims to supplement of exogenous bacteria,meanwhile,it is also regarded as a nutrient added into the environment.Up to now,how the microbial product influence the environment is poorly known.On the one hand,the exogenous bacteria may colonize and growth in the new environment,and then change the environment through the corresponding functional expression.On the other hand,the microbial product may affect the environment by affecting the native bacterial community and function without successfully colonization in the environment.In order to better understand the process and mechanism of microbial product in improving the aquaculture environment,we evaluated the efficiency of addition of microbial product,addition of carbon source and combined addition of microbial product and carbon source in improving the water quality of pond aquaculture based on in situ mesocosm and microcosm,and the responses of plankton community and transformation of inorganic nitrogen were investigated.The relationship between addition of microbial product and addition of carbon source was determined.The mainly results are listed as follows:1)Bacterial community and abiotic environmental parameters in twelve freshwater aquaculture ponds were analyzed.According to the major component of stocked animals,the ponds were grouped into four types:black carp Mylopharyngodon piceus,largemouth bass Micropterus salmoides,yellow catfish Pelteobagrus fulvidraco and pearl mussel Hyriopsis cumingii ponds.Each type of pond was stocked with three species of Chinese carps(silver carp,bighead carp and gibel carp)to form a unique mode of fish polyculture or mussel-fish integrated culture.The bacterial composition was identified by 16S rDNA sequencing.Totally 3701 and 11150 operational taxonomic units(OTUs)were identified from the water and sediment samples,respectively.The number of OTUs,abundance-based coverage estimator(ACE),Chaol index and Shannon diversity index were lower in the water column than in the sediment,suggesting that diversity and stability of bacterial community were higher in the sediment than in the water column.In the water column,Proteobacteria,Actinobacteria and Bacteroidetes dominated at the phylum level,and 26 dominant genera were identified.In the sediment,Proteobacteria,Chloroflexi,Bacteroidetes,Acidobacteria and Nitrospirae dominated at the phylum level,and 25 dominant genera were identified.Bacterial compositions between the ponds with different aquaculture modes were similar at the phylum levels,but varied at the genus levels.The bacterial composition in the ponds was correlated with hardness,ammonia,and total nitrogen in the water column.Though the relative abundance of ammonia oxidizing bacteria Nitrosomonadaceae_uncultured is higher in fish polyculture system than in mussel fish integrated culture,the activity of ammonia oxidizing bacteria failed to decrease the ammonia to a relatively low level in the fish polyculture ponds.Meanwhile,the relative abundance of nitrite oxidizing bacteria Nitrospinaceae_uncultured and concentration of nitrite were higher in the fish polyculture pond than in the mussel fish integrated culture.This study indicates that the type of aquaculture mode is a factor regulating the microbial community,which provides an insight towards microbial management through microbial manipulation in pond culture.2)An 80-day mesocosm experiment was conducted to evaluate the effect of four commercial microbial products on production performance and water quality in integrated culture of freshwater pearl mussel Hyriopsis cumingii,grass carp Ctenopharyngodon idellus and gibel carp Carassius gibelio.Five treatments were used.One treatment with non-supplementation of microbial products served as control(C).In the other four treatments,Novozymes Pond Protect(NO),Bio-Form BZT-Water Reform(WR),Bacillus natto(BN)and Effective Microbes(EM)were added at the intervals of two weeks,respectively.Mussel yield declined in the tanks with supplementation of the microbial products.No significant differences were found in chemical water quality among the treatments except total nitrogen was higher in tanks EM than in tanks C.Biomass of phytoplankton and Cyanophyta were higher in tanks NO,WR,BN and EM than in tanks C.Actinobacteria,Chlorobi,Verrucomicrobia and Proteobacteria dominated in the bacterial community,and supplementation of the microbial products resulted in change of their relatively abundance.Bacterial community in the tanks was significantly affected by total nitrogen,total phosphorus,chemical oxygen demand and Cyanophyta biomass.This study reveals that the function of these microbial products as probiotics is limited in H.cumingii farming.3)A 30-day experiment was conducted to evaluate the effect of C/N ratio on water quality and bacterial community in an integrated system comprising one molluscan species(pearl mussel Hyriopsis cumingii)and two species of fish(gibel carp Carassius gibelio and silver carp Hypophthalmichthys molitrix)at five C/N ratios(6,8,10,12 and 14).The mussel and fish were reared in the same tanks(400 L),but gibel carp received formulated feed.Water quality in the experimental tanks was analyzed on day 0,10,20 and 30,and bacterial community in the water column and sediment was analyzed on day 30.Total nitrogen,total phosphorus and total organic carbon accumulated in the tanks over time.Ammonia and nitrite decreased with the increase of C/N ratio.Bacterial community in the water column and sediment changed at the phylum and genus levels with the increase of C/N ratio,and the critical C/N ratio causing a functional shift of bacterial community occurred at 10 in water column and 12 in sediment.The increase of C/N ratio benefited the growth of both potential probiotics and pathogenic bacteria.The high C/N ratio enhanced the bacterial functions of chemoheterotrophy and hydrocarbon degradation,but depressed the functions of nitrification and denitrification in the water column and sediment,respectively.This study reveals that the C/N ratio can be used as a useful tool to manipulate the bacterial community and water quality in the mussel-fish integrated system.4)In this study,we compared the effect of supplement of carbon,microbial products and both carbon and microbial products on the water quality and bacterial community in a fish polyculture system.The experiment was performed with tanks which set in a mussel fish integrated aquaculture pond,and each tank included two grass carps Ctenopharyngodon idellus,two gibel carps Carassius gibelio and a silver carp Hypophthalmichthys molitrix.The grass carp and gibel carp were fed with a commercial feed daily.On the day 40,when the ammonia reached a relatively high level,the tanks divided into four groups,one group was regarded as control;one group was supplemented with glucose as carbon source(60 g per six day);one group was supplemented with two microbial products Novozymes Pond Protect and Novozymes Pond Plus(each 2 g per six day);one group was supplemented with both carbon source and microbial products,and each group has three replications.Ammonia,nitrite and nitrate decreased with the addition of carbon source,however the microbial products failed to improve the water quality.There was no interactive effect between the carbon and microbial product in decreasing the inorganic nitrogen.In addition,the bacterial community changed mainly attributed to the temporal change.Microbial products and carbon source have limited function in reshaping the bacterial community.The dominant taxa at the phylum and genus levels did not change by the treatment,whereas the microbial products and carbon source can affect the relative abundance of Bacillus and hgcI_clade,and interactive effect between microbial product and carbon source was detected.5)Phytoplankton growth and degradation are associated to the bacterial community in the phycosphere.The comparison of bacterial communities in two different competitive algal blooms(Microcystis aeruginosa and Euglena sanguinea)in an aquaculture pond provides a suitable way to better understand the variation in bacterial community structure and the role of bacteria in algal bloom formation.Membranes of three different pore sizes(10 ?m,1 ?m and 0.22 ?m in diameter)were used in succession to collect bacteria from water samples.The Proteobacteria and Bacteroidetes were the dominant phyla,whereas the relative abundance of Actinobacteria was low in the phycosphere of both blooms(E.sanguinea and M.aeruginosa).The percentage of Gemmatimonadetes was higher in large size(>10 ?m)aggregates while Bacteroidetes was higher in small size(1-10 ?m)aggregates,and the Candidate_division_TM7 was higher as free-living bacteria(0.22-1 ?m).Chlorobi,Chloroflexi and Fusobacteria were abundant in M.aeruginosa blooms water,whereas Deinococcus-Thermus was abundant in E.sanguinea blooms water.The functional group of chemoheterotrophy was mainly found in the small size aggregates and free living environment of both blooms.The functional groups of aerobic_ammonia_oxidation,nitrification and human_pathogens_all were mainly found in the large size aggregates of M.aeruginosa bloom.These results indicated that different size aggregates in the blooms provide different ecological niches where different bacterial functional groups adapted.In addition,the found of the specific bacterial communities associated with the M.aeruginosa and E.sanguinea blooms provide new insight of bacterial function in the bloom formation.6)Commercial microbial product of Novozymes pond plus and unialgal green algae Chlorella variabilis were chosen to determine the potential mechanism of exogenous bacteria in improving aquaculture water quality.The community respiration and gross primary productivity was observed in dark and light respectively,and the bacterial species in microbial product and the variation of bacterial composition in the algae medium were determined by 16S rDNA-based polymerase chain reaction-density gradient gel electrophoresis(PCR-DGGE)techniques.Through the 16S-rRNA sequence identified,two bacterial species(Bacillus licheniformis and Bacillus sp.)were detected as active bacteria added into the algae medium.The results showed that the community respiration increased significantly at the time of 0 hour to 17 hours in dark after the exogenous bacteria added,and the gross primary productivity strengthened significantly compared to the control in light.The bacteria and algae density in the group supplemented with microbial product increased significantly compared to the control.Whereas,53 hours later with the supplementation of exogenous bacteria,the exogenous bacteria were not detected and the bacteria community structure in the algae medium did not changed either.These results suggest that exogenous bacteria can improve the water quality by increasing the algae biomass and gross primary productivity without change the native bacterial community structure.7)The function of carbon source supplementation to control the inorganic nitrogen in polyculture system was determined in both mesocosm and microcosm.The mesocosms were constructed in 2000 L polyethylene tanks,and each tank included one-kilogram grass carp Ctenopharyngodon idellus,three gibel carp Carassius gibelio,one common carp Cyprinus carpio and one silver carp Hypophthalmichthys molitrix.The fish were fed with a commercial feed daily.After one month,the 18 tanks divided into three groups,one group was regarded as control,in the other two groups(LC and HC),the carbon source was supplemented based on the concentration of ammonia.In group LC,the carbon source was supplemented based on a ratio of ammonia/carbon at 1:3(3 g carbon source added for 1 g ammonia),In group HC,the carbon source was supplemented based on a ratio of ammonia/carbon at 1:6(6 g carbon source added for 1 g ammonia).The result indicated that the ammonia in all the groups decreased significantly,but their processes were different.The ammnia decreased in the control mainly attributed to the nitrification.The ammonia decreased in the group HC mainly attributed to the assimilation of microorganism.The ammonia decreased in the group LC mainly attributed to both nitrification and assimilation of microorganism.The distances within the groups in control and group HC were higher than that in group LC.The phylum Chloroflexi and genus Roseiflexus were sensitive to the C/N ratio,and their abundance increased with the increase of C/N ratio.In order to identify the role of bacteria and phytoplankton in controling the inorganic nitrogen,one microcosm was conducted using the 500 mL glass bottles in a fish polyculture tank.One group was regarded as control;one group was supplemented with glucose as carbon source;one group in which the phytoplankton was filtered out and then glucose was supplemented;One group in which the glucose was supplemented and the antibiotic was also added to inhibit the growth of bacteria.The result indicated that the bacteria and phytoplankton competitively utilized the ammonia as nitrogen source while the environment was rich in organic carbon.Bacteria could utilize the ammonia,nitrite and nitrate as nitrogen source.Howerver,the phytoplankton mainly utilized the ammonia.The assimilation of bacteria was depressed by the phytoplankton.The phylum Proteobacteria had advantage in competing with other phyla in the organic carbon rich environment,whereas the phyla Bacteroidete and Spirochaetae had disadvantage in competing with others in the organic carbon rich environment.The growth of Planctomycete,Acidobacteria and Firmicutes was depressed by the phytoplankton.8)The efficiency of carbon source and microbial product in controling the inorganic nitroge and their combined function were determined by an in-situ microcosm in a fish polyculture system.One group was regarded as control;One group was added with carbon source;One group was added with nitrifying bacteria;One group was added with heterotrophic bacteria;One group was added with both carbon and nitrifying bacteria.One group was added with both carbon and heterotrophic bacteria.The ammonia decreased in all the groups but their processes were different.In the control,the nitrite and nitrate increased with the decrease of ammonia.In the group added with carbon,only part of ammonia was transformed into nitrite.In the group added with microbial product or carbon source or both microbial product and carbon source,the ammonia decreased while the nitrite and nitrate decreased at the same time,and the interactive effect between the microbial product and carbon was found.The nitrifying bacteria did not show the function of nitrification except on day 2.All the microbial products showed the function of nitrogen assimilation in decreasing the inorganic nitrogen.In order to futher indentify whether the microbial product has potential carbon effect,the efficiency in controlling the inorganic nitrogen was compared between the microbial product and the sterilized microbial products in another in situ microcosm.The result showed that the inorganic carbon and organic carbon increased with the supplementaton of microbial product or sterilized microbial product.The ammonia kept stable in the control,while the ammonia firstly decreased and then increased in both the microbial product treatment and sterilized microbial product treatment.The nitrite kept stable in the control,while the nitrite firstly increased and then decreased in both the microbial product treatment and sterilized microbial product treatment.The nitrate kept stable in the control,while the nitrate decreased in both the microbial product treatment and sterilized microbial product treatment.The microbial product and sterilized microbial product showd the similar function as poential carbon effect in controlling the inorganic nitrogen.The result also indicated that the efficiency of the carbon effect in controlling the inorganic nitrogen highly depended on the backgound of the environment.We can draw several conclusions from the results:1)The microbial product of Novozymes Pond Protect,Bio-Form BZT-Water Reform,Bacillus natto and Effective Microbes had limited role in improving the water quality and yield of integrated culture of freshwater pearl mussel Hyriopsis cumingii,grass carp Ctenopharyngodon idellus and gibel carp Carassius gibelio.2)The addition of exogenous carbon could improve the water quality of integrated culture of freshwater pearl mussel Hyriopsis cumingii,grass carp Ctenopharyngodon idellus,gibel carp Carassius gibelio and silver carp Hypophthalmichthys molitrix or polyculture culture of grass carp Ctenopharyngodon idellus,gibel carp Carassius gibelio,common carp Cyprinus carpio and silver carp Hypophthalmichthys molitrix.Besides,the bacterial community both in the water column and sediment could be influenced by the carbon nitrogen ratio.3)The comparison of function between the sterilized microbial product and unsterilized microbial product indicated that the microbial product might play a role regarded as carbon and influenced the transformation of inorganic nitrogen.