Fish waste management by conversion into heterotrophic bacteria biomass

Just as all other types of animal production, aquaculture produces waste. This waste can be managed outside the production system, comparable to terrestrial husbandry systems. However, particularly recirculation aquaculture systems (RAS) are suited to manage waste within the system. In this case, processes have to be selected to convert the waste into a re-usable product. Dissolved and solid waste conversion by heterotrophic bacteria is one of these processes. In the present study, the potential of the latter process was investigated. An operational scheme was followed, which contained five steps: (1) to evaluate nutrient flows in integrated aquaculture systems, (2) to select and to investigate a conversion process, (3) to improve the process and analyze its sensitivity, (4) to evaluate the product suitability, (5) to derive the kinetics, reactor design, and to determine the integration possibilities into RAS.In chapter 2 nutrient flows, conversions and waste management were evaluated, which are taking place in integrated intensive aquaculture systems. In these systems, fish is cultured next to other organisms, which are converting nutrients, which would be otherwise discharged. These conversions were evaluated based on nitrogen (N) and phosphorous (P) balances using a mass balance approach. In the reviewed examples, fish culture alone retained 20-50% feed N and 15-65% feed P. The combination of fish culture with phototrophic conversion increased nutrient retention of feed N by 15-50% and of feed P by up to 53%. If in addition herbivore consumption was included, then the gained nutrient retention decreased by 60-85% feed N and 50-90% feed P. The conversion of nutrients into bacteria and detrivorous worm biomass contributed only to a smaller extent (e.g. 7% feed N and 6% feed P and 0.06% feed N 0.03xl0"3% feed P, respectively). AIl integrated modules had their specific limitations, which were related to uptake kinetics, nutrient preference, unwanted conversion processes and abiotic factors and implications.Chapters 3 to 5 focused on the experimental production of heterotrophic bacteria biomass on carbon (C) supplemented fish waste under different operational conditions. The results covered step two and three in the operational scheme.In chapter 3, the drum filter effluent from a RAS was used as substrate to produce heterotrophic bacteria in suspended growth reactors. Effects of organic C supplementation (0, 0.9, 1.7, 2.5gC/l as sodium acetate) and of hydraulic retention times (HRT: 1 l-lh) on bacteria biomass production and nutrient conversion were investigated. Bacteria production, expressed as VSS (volatile suspended solids) was enhanced by organic C supplementation, resulting in a production of 55-125gVSS/kg fish feed (0.2-0.5gVSS/gC). Maximum observed crude protein production was ~100g protein/kg fish feed. The metabolic maintenance costs were 0.08Cmol/Cmol h"', and the maximum growth rate was 0.25-0.5h"'. Approximately, 90% of the inorganic nitrogen and 80% of ortho-phosphate-phosphorus were converted.The influence of nitrogenous waste on bacteria yields was investigated in chapter 4. RAS effluents are rich in nitrate and low in total ammonia nitrogen (TAN). This might result in 20% lower bacteria yields, because nitrate conversion into bacteria is less energy efficient than TAN conversion. In this chapter, the influence of TAN concentrations (1, 12, 98, 193, 257mgTAN/l) and stable nitrate-N concentrations (174+29mgfl) on bacteria yields and N conversions was investigated in a RAS under practical conditions. The effluent slurry was supplemented with 1.7gC71 sodium acetate, due to C deficiency, and was converted continuously in a suspended bacteria growth reactor (6h HRT). TAN utilization did not result in different yields compared to those for nitrate (0.24-0.32gVSS/gC, p=0.763). However, TAN was preferred compared to nitrate and was converted to nearly 100%, independently of TAN concentrations. TAN and nitrate conversion rates differed significantly for increasing TAN levels (p<0.000 and p=0.012), and were negatively correlated. It seems, therefore, equally possible to supply the nitrogenous substrate for bacteria conversion as nitrate or as TAN. Because in RAS, nitrate is the predominant N form in the waste, the bacteria reactor can safely be integrated into an existing RAS as end of pipe treatment.In chapter 5, sodium acetate, which was used in chapter 3 and 4 was replaced by molasses as organic C supplement. The effect of molasses as alternative C source on bacteria productions and yields was investigated. One bacteria reactor (3.5 1) was connected to the drum filter (filter mesh size 60???) outlet of a recirculation system in a continuous flow (HRT: 6h). The different supplementation levels of molasses were 0.0, 3.2, 5.8, 7.8, 9JgCfl/d. For the maximum flux, the VSS and crude protein production were about 168gVSS and 95g crude protein per kg feed. The maximum conversion of nitrate and ortho-phosphate was 24g NO3-N and 4gP/kg feed, a conversion of 90% of the inorganic nitrogenous waste and 98% of the ortho-phosphate-P. Furthermore the maximum substrate removal rate and the half saturation constant (K8) were determined (1.62gC/l^i and 0.097gC/l respectively). The maximum specific removal rate was 0.31gC/gVSS/h and the related K5 was 0.008gC/l. The observed growth rate reached a maximum for C fluxes higher than 8g/l/d.Chapter 6 and 7 were focusing on the fourth step of the operational scheme (product evaluation and determination of re-use potential).Because the produced bacteria biomass might contain pathogens, which could reduce its suitability as feed, it was important to characterize the obtained bacteria communities under different conditions (chapter 3 to 5, reported in chapter 6). The operation conditions were: 7h hydraulic retention time versus 2h, sodium acetate versus molasses (organic C supplement), and ammonia versus nitrate (N donor). Samples were analyzed by standard biochemical tests, by 16sRNA ribotyping and ribosomal RNA gene-targeted PCR-DGGE fingerprinting combined with clone library analysis. The community of the drum filter effluent was different from the communities found in the bacteria reactors. However, all major community components were present in both the drum filter effluent and reactor broths. HRTs (7h versus 2h) influenced bacteria community resulting in a more abundant fraction of alpha proteobacterium Bioluz/ Acinetobacter at 2h HRT compared to 7h HRT (Rhizobium! Mezorhizobium). The use of molasses instead of sodium acetate changed the bacteria community from Rhizobium/ Mesorhizobium to Aquaspirillum as major component. Providing TAN in addition to nitrate as nitrogenous substrate led to the occurrence of bacteria close to Sphaerotilus, Sphingobacterium and Jonesia. From those results, it was concluded that 6-7h HRT is recommended, and that the type of substrate (sodium acetate or molasses, TAN or nitrate) is less important, and results in communities with a comparable low pathogenic risk.In chapter 7, the produced bacteria biomass was fed to shrimps (Litopenaeus vannamei), In total three different diets were used in a variance of a T-maze test: a commercial shrimp feed, the bacteria biomass, which was produced in the suspended growth reactors on C supplemented fish waste under conditions, comparable to those reported in chapter 3, and slurry, which was anaerobicaliy produced in a denitrification reactor. If the bacteria products would be attractive as diet, the nutrient retention of the RAS would be improved, resulting in a system, combining fish, bacteria and shrimp. The diet preference was interpreted as an expression of diet attractiveness. As a first result, shrimp were moving from an equal distribution before feeding (+/-50%, -2min), towards the feeding places (>50%, 2, 5, and 10 minutes after feeding). It was, therefore, inferred, that all bacteria biomass and commercial feed combinations were basically attractive for the shrimp. This response was not instantaneous. After feeding (2min) more than 80% of the shrimp were present at the feeding places and showed a significant preference for the commercial feed compared to the aerobically produced bacteria slurry. For the other diet combinations no significant differences could be detected for 2min. For 5 and lOmin after feeding, shrimp behavior changed from the commercial feed to the aerobically and anaerobicaliy produced bacteria biomass segments. From this study it was conc!uded that although the commercial diet was preferred above the aerobic slurry, the bacteria slurries had also attracted the shrimps. There was no unambiguous conclusion to be made regarding the preference for aerobic or anaerobic produced slurry. In chapter 8, the design of a suspended bacteria growth reactor integrated in a lOOMT African catfish farm was determined. This study integrated results from the earlier chapters to calculate the bacteria kinetics (yield=0.537gVSS/gC; endogenous decay coefficienl=0.033h^'; maximum specific growth rate=0.217h^ ; half-velocity constant=0.025g/l; and maximum rate of substrate utilization-0.404gC/gVSS*h). As part of the study a model was developed and validated. This model was used to calculate the VSS production and nutrient conversion by heterotrophic bacteria conversion for a lOOMT African catfish farm. The VSS production was 187gVSSAcg feed and the inorganic nutrients (N and P) were removed with an efficiency of 85 and 95% for a C supplementation level of 3.5gC/l (455gC/kg feed). A reactor integrated in a lOOMT farming facility would have a volume of 11m , based on a minimum HRT of6h. The production and potential re-use of heterotrophic bacteria biomass is, therefore, a prospective tool to lower nutrient discharge and to increase nutrient retention and sustainability of RAS in the future.