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In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities. Bacteria were produced in growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs. 2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the drumfilter effluent was different from those found in the reactors. However, all major community components were present in the effluent and reactor broths. HRT influenced the bacteria community, resulting in a DGGE profile dominated by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT compared to 7 h HRT, where bands indicative of alpha-proteobacterial populations most closely related to Rhizobium and Shinella spp. were most abundant. Molasses influenced the bacterial community. It was dominated by an Aquaspirillum serpens-related population. Providing total ammonia nitrogen (TAN) in addition to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacterium mizutaii and Jonesia spp. It was concluded from these results that a 6-7 h HRT is recommended, and that the type of substrate is less important, and results in communities with a comparably low pathogenic risk.

The kinetics and design of a suspended bacteria growth reactor, which can be integrated in a 100MT African catfish farm, were determined. Such a reactor converted nitrogen (N) and phosphorus (P) from RAS effluents into heterotrophic bacteria biomass. The determined kinetics were: yield = 0.537 g VSS/g C; endogenous decay coefficient = 0.033 h −1 ; maximum specific growth rate = 0.217 h −1 ; half-velocity constant = 0.025 g/l; maximum rate of substrate utilization = 0.404g C/g VSS h. A reactor integrated in a 100MT farming facility would have a volume of 11 m 3 , based on a minimum HRT of 6 h. The kinetics and reactor design were integrated in a model to predict the VSS production (volatile suspended solids as measure of bacteria biomass) and nutrient conversions. The VSS production was on average 187 ± 2 g VSS/kg feed and the inorganic nutrients (N and P) were removed with an efficiency of 85 ± 3.0% and 95 ± 2.5%, respectively. A carbon (C) supplementation level of 455 g C/kg feed was required to ensure optimal C:N ratios for heterotrophic bacteria production. The production 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.

This paper analyses nutrient conversions, which are taking place in integrated intensive aquaculture systems. In these systems fish is cultured next to other organisms, which are converting otherwise discharged nutrients into valuable products. These conversions are analyzed based on nitrogen and phosphorous balances using a mass balance approach. The analytical concept of this review comprises a hypothetical system design with five modules: (1) the conversion of feed nutrients into fish biomass, the “Fish-Biomass-Converter”; (2) the separation of solid and dissolved fish waste/nutrients; the “Fish-Waste-Processor”; (3) the conversion of dissolved fish waste/nutrients, the “Phototrophic-herbivore-Converter”; (4 and 5) the conversion of solid fish waste, the “Bacterial-Waste-Converter”, or the “Detrivorous-Converter”. In the reviewed examples, fish culture alone retains 20–50% feed nitrogen (N) and 15–65% feed phosphorous (P). The combination of fish culture with phototrophic conversion increases nutrient retention of feed N by 15–50% and feed P by up to 53%. If in addition herbivore consumption is included, nutrient retention decreases by 60–85% feed N and 50–90% feed P. This is according to the general observation of nutrient losses from one trophic level to the next. The conversion of nutrients into bacteria and detrivorous worm biomass contributes only in smaller margins (e.g. 7% feed N and 6% feed P and 0.06% feed N 0.03 × 10−3% feed P, respectively). All integrated modules have their specific limitations, which are related to uptake kinetics, nutrient preference, unwanted conversion processes and abiotic factors.

Cellulose fatty esters were prepared by acylation of cellulose acetate with various fatty acids catalyzed by immobilized lipase from Candida antarctica (Novozyme) in acetonitrile. The reaction parameters affecting the biocatalytic process such as the role of the nature of the substrates, the concentration of the enzyme and the reactants as well as the agitation speed of the reaction mixture have been investigated. At optimum reaction conditions about 35% of the initial amount of fatty acid were incorporated on the modified cellulose acetate molecule, while an increase of 2% of the ester bonds was observed on the final product. Journal of Biotechnology

The acylation of glucose with lauric acid in a reaction catalysed by two Candida lipases and a Mucor miehei lipase in supercritical carbon dioxide (SCCO2) was investigated. A linear dependence of the reaction rate on enzyme concentration was observed. Studies on the effect of temperature on enzyme activity showed that Candida antarctica lipase remains stable at temperatures as high as 70Β°C. Non-immobilised Candida rugosa lipase was found to have a temperature optimum at 60Β°C. The acylation reaction rate depended on the initial water activity of both substrates and enzyme; the optimum was 0Β·75 for Candida antarctica lipase, 0Β·53 for Candida rugosa lipase, and between 0Β·3 and 0Β·5 for Mucor miehei lipase. Candida rugosa lipase was most active at a molar ratio of sugar: acyl donor of 1: 3, while the optimum ratio was found to increase to 1: 6 when the reaction was catalysed by Candida antarctica and Mucor miehei lipases. Β© 1998 SCI Journal of Chemical Technology & Biotechnology

Cutinase from Fusarium solani pisi was immobilized on Accurel EP100 and catalyzed the esterification of hexanoic acid with hexanol in supercritical CO. The enzyme lost only 10% of its activity over six days. Esterification was maximal with a water activity of a = 0.76.

The drumfilter effluent from a recirculation aquaculture system (RAS) can be used as substrate for heterotrophic bacteria production. This biomass can be re-used as aquatic feed. 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 study the influence of TAN concentrations (1, 12, 98, 193, 257 mg TAN/l) and stable nitrate-N concentrations (174 ± 29 mg/l) on bacteria yields and nitrogen conversions was investigated in a RAS under practical conditions. The effluent slurry was supplemented with 1.7 g C/l sodium acetate, due to carbon deficiency, and was converted continuously in a suspended bacteria growth reactor (hydraulic retention time 6 h). TAN utilization did not result in significantly different observed yields than nitrate (0.24¿0.32 g VSS/g C, p = 0.763). However, TAN was preferred compared to nitrate and was converted to nearly 100%, independently of TAN concentrations. TAN and nitrate conversions rates were differing significantly for increasing TAN levels (p

The effluent from the drumfilter of a recirculation aquaculture system was used as substrate to produce heterotrophic bacteria in suspended growth reactors. The effects of organic carbon supplementation (0, 3, 6, 8 g/l sodium acetate) and of hydraulic retention times (11-1h) on bacteria biomass production and nutrient conversion were investigated. Bacteria production, expressed as volatile suspended solids (VSS), was enhanced by organic carbon supplementation, resulting in a production of 55-125 g VSS/kg fish feed (0.2-0.5 g VSS/g carbon). Maximum observed crude protein production was approximately 100 g protein/kg fish feed. The metabolic maintenance costs were 0.08 Cmol/Cmol h, and the maximum growth rate was 0.25-0.5 h(-1). Ninety percent of the inorganic nitrogenous and 80% of ortho-phosphate were converted. Producing bacteria on the drumfilter effluent results in additional protein retention and lowers overall nutrient discharge from recirculation aquaculture systems.

The drumfilter effluent from a recirculation aquaculture system (RAS) can be used as substrate for heterotrophic bacteria production. These bacteria can be reused as aquatic feed. In RAS drumfilter effluents are organic carbon deficient for bacteria production. This is due to nitrogen accumulation in the system water. In the present experiment, a bacteria growth reactor (3.5l) was connected to the drumfilter (filter mesh size 60 μm) outlet of a recirculation system. To counteract carbon deficiency, different supplementation levels of molasses (organic carbon) were tested (carbon fluxes of 0.0, 3.2, 5.8, 7.8, 9.7 gC molasses/l/d; C:N ratios: 3.4, 6.4, 9.4, 13.0 and 16.5). The hydraulic retention time in the bacteria reactor was 6 h. For the maximum flux, the VSS and crude protein productions were about 168 gVSS and 95 g crude protein per kg feed. The maximum conversion of nitrate and ortho-phosphate was 24 g NO3−N and 4 gP/kg feed. This equals a conversion of 90% of the inorganic nitrogenous waste and 98% of the ortho-phosphate–P. Furthermore the maximum substrate removal rate and the Ks for molasses were determined (1.62 gC/l/h and 0.097 gC/l respectively). The maximum specific removal rate was 0.31 gC/gVSS/h and the related half saturation constant was 0.008 gC/l. The observed growth rate reached a maximum for C fluxes higher than 8 g/l/d. The present integration of heterotrophic bacteria production in RAS represents, therefore, an innovative option to reduce waste discharge by conversion. If the bacteria are reused as feed, system's ecological sustainability increases.

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8-12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (aw) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis-Menten kinetics. Journal of Molecular Catalysis B: Enzymatic

Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%.

The enzymatic esterification of natural phenolic antioxidants such as cinnamic acid and benzoic acid derivatives, with aliphatic alcohols, monosaccharides as well as alkylglucosides, using various lipases and esterases in non-aqueous media, was investigated. Reaction rate and esterification yield seems to be linked to the structural characteristics of the substrates (aromatic acids and alcohols or sugars) used. Journal of Molecular Catalysis B: Enzymatic

Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%. Biotechnol Bioeng

The regioselective acylation of fructose with various fatty acids catalyzed by immobilized lipases from Mucor miehei and Candida antarctica in tert-butanol was investigated. The reaction parameters affecting the catalytic activity as well as the regioselectivity of the reaction, such as the nature and the concentration of the acyl-donor and the water activity (aw) of the system, were studied. Yields above 50 % of ester were obtained after 72h of incubation when the reaction was carried out with an excess of fatty acid.

The esterification of some natural antioxidants such as cinnamic acid derivatives and ascorbic acid in non-aqueous media, catalyzed by immobilized lipases from Candida antarctica and Rhizomucor miehei , was investigated. The alcohol chain length affected the rate of esterification of cinnamic acids by both lipases. Higher reaction rates were observed when the esterification was carried out with medium- or long-chain alcohols. The rate also depended on aromatic acid structure. The reactivity of the carboxylic function of the cinnamic acids was affected by electron-donating substituents in the aromatic ring. Higher yields were observed for the esterification of p -hydroxyphenylacetic acid (97%) catalyzed by C. antarctica lipase and for the esterification of cinnamic acid (59%) catalyzed by R. miehei lipase. Candida antarctica lipase was more suitable for producing ascorbic acid fatty esters, catalyzing with a relatively high yield (up to 65% within 24 h) the regioselective esterification of ascorbic acid with various fatty acids in 2-methyl-2-propanol. The reaction rate and yield depended on the fatty acid chain length and on the molar ratio of reactants. All ascorbic acid fatty esters produced by this procedure exhibited a significant antioxidant activity in a micellar substrate composed of linoleic acid. Journal of the American Oil Chemists' Society

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 (p50%, 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.