Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l⁻¹ after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

K88大肠杆菌菌毛蛋白发酵工艺条件优化研究

分别采用LB培养基、牛肉膏蛋白胨培养基、强化营养培养基、玉米浆培养基对大肠杆菌K88进行发酵培养,选出最适于大肠杆菌K88生长的玉米浆培养基;采用正交实验对玉米浆培养基的C／N、K2HPO4／KH2PO4、Mg^2＋的配比进行优化,筛选最适于大肠杆菌K88生长的营养配比;研究生长曲线、接种量以及菌体和菌毛生产量的相关性,根据实验结果优化发酵培养条件,确定菌种的最佳发酵工艺,以收获最多的K88菌毛蛋白。研究表明,K88大肠杆菌在玉米浆培养基C／N、K2HP04／KH2PO4的配比分别为5／11、1／1,Mg^2＋为0．1g/mL,pH值为7．2,转速为200r／min,接种量为4．5％的条件下发酵26个小时,菌体和菌毛生产量均达到高峰,同时得出菌毛蛋白产生量和菌体量成正相关。     

Operation strategies for biohydrogen production with a high-rate anaerobic granular sludge bed bioreactor

Long-term operation for biohydrogen production with an efficient carrier-induced granular sludge bed (CIGSB) bioreactor had encountered problems with poor biomass retention at a low hydraulic retention (HRT) as well as poor mass-transfer efficiency at a high HRT or under a prolonged operation period. This work was undertaken to develop strategies enabling better biomass retention and mass-transfer efficiency of the CIGSB reactors. Supplementation of calcium ion was found to enhance mechanical strength of the granular sludge. Addition of 5.4–27.2 mg/l of Ca²⁺ also led to an over three-fold increase in biomass concentration and a nearly five-fold increase in the H₂ production rate (up to 5.1 l H₂/h/l). Two reflux strategies were utilized to enhance the mass-transfer efficiency of the CIGSB system. The liquid reflux (LR) strategy enhanced the H₂ production rate by 2.2-fold at an optimal liquid upflow velocity of 1.09 m/h, which also gave a maximal biomass concentration of ca. 22 g VSS/l. Similar optimal H₂ production rate was also obtained with the gas reflux (GR) strategy at a rate of 1.0–1.49 m/h, whereas the biomass concentration decreased to 2–7 g VSS/l and thereby the specific H₂ production rate was higher than that with LR. The operation strategies applied in this work were effective to allow stable and efficient H₂ production for nearly 100 days.     

漏斗多孔菌液体菌种培养基及其栽培条件

为实现漏斗多孔菌资源化利用,设计单因素试验,以菌丝生物量、菌球密度和菌球直径为指标,获得漏斗多孔菌Polyporus arcularius液体菌种培养基配方为马铃薯（去皮）200g、玉米粉20.0g、蛋白胨5.0g、KH₂PO₄ 3.0g、K₂HPO₄ 1.0g,MgSO₄·7H₂O 1.5g,初始pH 5.0,并优化培养条件为装液量250mL/500mL,接种量2%,培养温度32℃,转速170r/min,种龄6d;依上述条件制备液体菌种,以固体菌种为对照,进行出菇试验,调查农艺性状,试验结果表明液体菌种接种后,菌袋满袋时间提前7d,子实体原基产生时间提前8d,头潮菇鲜重提高22.5%;配方试验结果表明,以细木屑为主料的配方产量最高,头潮菇鲜重为270.6g/袋。     

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H₂O₂ was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1–100 μg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1–10 μg/ml. At a concentration of 100 μg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H₂O₂ at concentrations of 0.1–10 μg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H₂O₂. All the compounds at 10 μg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H₂O₂ in vitro.     

A Holocene environmental record from the southern Yangtze River delta, eastern China

The Holocene sedimentary record of core ZX-1, recovered west of mid-Holocene Chenier ridges on the monsoon-controlled Southern Yangtze delta, eastern China, consists of lagoon, salt marsh, upper tidal flat, and limnic facies, reflecting low-energy depositional environments. Holocene deposits mainly originated from the surrounding Tai Lake drainage basin. The temporal variation of most geochemical element percentages corresponds with the climatic phases inferred from the pollen record, i.e., the relatively low values of SiO₂, Na₂O, CaO and high values of Al₂O₃, K₂O, MgO, Fe₂O₃, FeO, FeO + Fe₂O₃ and TiO₂ generally concur with the warm and humid climate, and vice versa. Three geochemical indices − Al₂O₃/Na₂O, K₂O/Na₂O, and CaO/K₂O − are found to be sensitive to past precipitation in the monsoon-controlled southern Yangtze River delta. Based on fine sediment analysis of core ZX-1, the samples deposited under a dry climate with a mean annual precipitation (MAP) < 500–900 mm tend to have Al₂O₃/Na₂O values less than 12.2, and the samples deposited under a moist climate with an MAP 1000–1800 mm mostly have Al₂O₃/Na₂O values higher than 12.2. Similarly, the K₂O/Na₂O boundary value is 2.0. However, for CaO/K₂O, the dry climate sediments likely show values higher than 0.9, and those wet climate ones generally have values less than 0.9. This geochemical response suggests the potential application of these indices in the interpretation of palaeoclimate variation of monsoon-controlled eastern China.

Holocene climatic variation history is reconstructed for the southern Yangtze delta. From 8000 to 7000 yr BP, regional climate demonstrated frequent fluctuations, with warm and wet periods (8000–7700; 7500–7200 yr BP) alternating with cool and less humid periods (7700–7500; 7200–7000 yr BP). From 7000 to 6000 yr BP, the climate was relatively warm and humid. Since then, it had turned cool and dry, climaxing in an intense cold event around 4000 yr BP. After the cold event, it became warm and humid until around 2500 yr BP.     

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H₂) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H₂ constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H₂ producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H₂ – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium.     

Metal–salt co-tolerance and metal removal by indigenous cyanobacterial strains

Chromium and salt tolerance in five indigenous cyanobacterial strains isolated from contaminated sites was investigated along with their metal bioaccumulative potential. All the five species showed significantly better growth when the medium was spiked with salt or chromium. As compared to single metal or salt treatment, the binary metal–salt (MS) treatments had more favorable effect on cyanobacterial growth as indicated by significantly higher concentration of the primary photosynthetic pigment chlorophyll at M₂₀S₂₀₀₀ (9.9–25.3 μg/mL) as compared to that at M₀S₀ (4.0–12.3 μg/mL). Similarly biomass was much higher at M₂₀S₁₀₀₀ and M₂₀S₂₀₀₀ (41.8–86.2 mg/10 mL) as compared to that at control, M₀S₀ (21.5–36.3 mg/10 mL). Accessory pigments like carotenoids and phycobilinproteins too tended to increase significantly in response to both metal and salts in the two species of Lyngbya (L. putealis and L. ceylanica var. constricta) and Gloeocapsa. These species also showed greater potential of chromium bioaccumulation, which increased further as both salt and metal concentration increased. In the two species of Nostoc however, bioaccumulative potential improve at higher metal concentration, but not affected significantly by salt concentration.     

Influence of inoculum activity on the bio-methanization of a kitchen waste under different waste/inoculum ratios

The use of a granular inoculum prevented acidification during the anaerobic batch biodegradation of a kitchen waste for waste/inoculum ratios in the range of 0.5–2.3 g VS/g VS, when the alkalinity/COD ratio was 37 mg NaHCO₃/g COD. In similar experiments but using a suspended sludge with a significantly lower activity, the methane production rates and the biodegradability were significantly lower and the pH decreased below 5.5 at the waste/inoculum ratio of 2.3 g VS/g VS. When the added alkalinity was decreased to 2 mg NaHCO₃/g COD, the ratio waste/inoculum was clearly more important than the inoculum activity, since, irrespective of the sludge used, acidification occurred at waste/inoculum ratios higher than 0.5 g VS/g VS. The advantage of using granular sludge was further investigated in order to define reasonable condition of waste/inoculum ratio and added alkalinity that could be applied in practice. For a waste/inoculum ratio of 1.35, there were no significant differences between the results obtained for the biodegradability and maximum methane production rate (MMPR), when the alkalinity decreased from 44 to 22 mg NaHCO₃/g COD.     

In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon

Esenbeckia febrifuga (Rutaceae) is a plant traditionally used to treat malaria in the Brazilian Amazon region. Ethanol extract of stems displayed a good antiplasmodial activity against Plasmodium falciparum strains W-2 (IC₅₀ 15.5±0.71 μg/ml) and 3 D7 (IC₅₀ 21.0±1.4 μg/ml). Two coumarins (bergaptene 1 and isopimpinellin 2), five alkaloids (flindersiamine 3, kokusaginine 4, skimmiamine 5, γ-fagarine 6 and 1-hydroxy-3-methoxy-N-methylacridone, 7), besides a limonoid (rutaevine 8), have been isolated for the first time from this species. Antiplasmodial activity of compounds 3, 5–8 has been evaluated in vitro against P. falciparum strains (W-2 and 3D7) and the furoquinolines 5 and 6 were the most potent displaying IC₅₀ values <50 μg/ml; flindersiamine (3) showed a weak activity while alkaloid 7 and rutaevine (8) were inactive (IC₅₀>100 μg/ml).     

Pretreatment of beet molasses to increase pullulan production

Pretreatment of beet molasses with cation exchange resin, sulphuric acid, tricalcium phosphate, potassium ferrocyanide, and ethylenediaminetetraacetic acid and disodium salt (EDTA) to increase the production of pullulan was investigated. Among the above techniques used for the removal of heavy metals, sulphuric acid treatment gave better results regarding polysaccharide concentration, polysaccharide yield, and sugar utilization. Aureobasidium pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The pullulan content of the crude polysaccharide was 30–35%. The addition of nutrients improved the production of polysaccharide. A maximum polysaccharide concentration (32·0±1·0 g litre⁻¹) was achieved in molasses solution (70 g litre ¹ initial sugar concentration, pH 6·5–7·5) treated with sulphuric acid and supplemented with K₂HPO₄ 0·5%, -glutamic acid 1%, olive oil 2·5% and Tween 80 0·5%. In this case, the highest values of biomass dry weight (33·8±1·0 g litre⁻¹), polysaccharide yield (63·5±2·5%), and sugar utilization (97·5±1·5%) were obtained at pH 6·5, 3·5, and 4·5–7·5, respectively.     

高产纤维素酶的拟康宁木霉菌株8985固态发酵条件优化

木霉是重要的产纤维素酶真菌,在其可利用性评价筛选过程中,获得了一株在实验室条件下高产纤维素酶的拟康宁木霉菌株8985。采用响应面法对8985产纤维素酶的固态发酵条件进行了研究,以滤纸酶活为响应值,通过Plackett-Burman设计对11个因素进行了筛选,包括温度、湿度、发酵时间、K₂HPO₄、(NH₄)₂SO₄、Tween-80、麸皮添加量、CaCl₂、MgSO₄、乳糖和pH;分析结果表明,影响产酶的3个关键因素分别为发酵时间、K₂HPO₄浓度和麸皮添加量。进而采用最陡爬坡试验逼近3个关键因素的最大响应区域,采用Box-Behnken设计建立数学模型,获得最佳产酶条件为：发酵5d、K₂HPO₄ 5.18g/L、麸皮添加量0.281g/g,滤纸酶活性可达7.00IU/g,与理论值7.03IU/g接近,是优化前的1.37倍,是菌株里氏木霉QM9414该酶活的2.92倍。研究结果为木霉菌种资源的利用提供了有价值的科学信息。     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Two new multi-cobalt-containing heteropolyoxotungstates

Two new multi-cobalt-containing polyoxotungstates K₄Na₆Co₂(H₂O)₁₂{Co(H₂O)₄[Co₂(H₂O)₁₀Co₄(H₂O)₂(B--SiW₉O₃₄)₂]₂} · 40H₂O (1) and K₁₀Na₂[Co₄(H₂O)₂(GeW₉O₃₄)₂] · 20H₂O (2) have been obtained by the routine synthetic reactions in aqueous solution. The polyoxoanion framework of 1 consists of two sandwich-type polyoxoanions [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ connected together by a [CoO₂(H₂O)₄] cluster to constitute the sandwich dimer, and then, four isolated Co(H₂O)₅ cations coordinate to the dimer through four μ₂-O atoms. The polyoxoanion 2 is isomorphic to the sandwich-type polyoxoanion [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ in 1. The magnetic property of compound 1 has been studied by measuring its magnetic susceptibility in the temperature range 2.0–300.0 K, indicating the existence of intramolecular ferromagnetic Co–Co interactions, and, the electrochemical properties of 1 and 2 are detected in the pH 4 buffer solution.     

营养盐初始组合浓度对类波氏真眼点藻生长和油脂积累的影响

为了研究培养基中主要营养元素氮、磷、硫初始组合浓度对类波氏真眼点藻(Eustigmatos cf.polyphem D.J.Hibberd)生长、油脂积累和脂肪酸组成的影响,分别以NaNO_3、K_2HPO_4、MgSO_4为氮、磷、硫源,设置不同的营养盐初始组合浓度对其进行培养,采用干重法、重量法、气相色谱分析法、元素分析仪等依次对生物量、总脂、脂肪酸组成和细胞内元素含量进行测定。结果显示,氮、磷、硫三者的初始组合浓度变化对类波氏真眼点藻的生长和油脂积累具有明显的影响,9 mmol/L NaNO_3、0.92 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4营养条件下其生物量达到最大值,为9.19 g/L;低氮、低磷胁迫或二者共同胁迫均促进其油脂积累,但低硫胁迫对油脂的积累影响不大,最大油脂含量出现在3 mmol/L NaNO_3、0.029 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4条件下,为68.7%(DW)。此外,类波氏真眼点藻富含棕榈油酸,占总脂肪酸含量的最大值达60.88%。选择合理的氮、磷、硫初始组合浓度能够有效促进其生长或油脂及棕榈油酸的积累。     

Synthesis and anticancer evaluation of certain indolo[2,3-b]quinoline derivatives

The present report describes the synthesis and anticancer evaluation of certain 11-substituted 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 6H-indolo[2,3-b]quinoline derivatives 11–13 were prepared from the commercially available 1,4-dihydroxyquinoline through alkylation, chlorination, nucleophilic reaction, and ring cyclization. Depending on the ratio of 11, (MeO)₂SO₂, and K₂CO₃, alkylation occurred primarily on N-5 (1:0.8:0.8) or N-6 (1:1.5:1.5) leading to the isolation of 14a or 14b as a major product. Accordingly, major product 15a (2/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 15b (1:1:1), respectively, was obtained by alkylation of 12 while 16a (13/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 16b (1:1:1), respectively, was obtained by alkylation of 13. The in vitro anticancer assay indicated 5-methylated derivatives 14a, 15a, 16a are more cytotoxic than their respective 6-methylated counterparts 14b, 15b, 16b and 6H-indolo[2,3-b]quinoline precursors 11, 12, 13. Among them, 11-(4-methoxyanilino)-6-methyl-6H-indolo[2,3-b]quinoline (16a) was the most cytotoxic with a mean GI₅₀ value of 0.78 μM and also exhibited selective cytotoxicities for HL-60 (TB), K-562, MOLT-4, RPMI-8226, and SR with GI₅₀ values of 0.11, 0.42, 0.09, 0.14, and 0.19 μM, respectively.     

Hydrogenotrophic denitrification of synthetic aquaculture wastewater using membrane bioreactor

A hydrogenotrophic denitrification system was evaluated in removing nitrate from synthetic aquaculture wastewater for recirculation purposes. Two membrane bioreactor (MBR) systems, namely, aeration–denitrification system (ADS) and denitrification–aeration system (DAS) were studied with 50 mg/L of influent concentrations for both organic matter and nitrate nitrogen. The DAS achieved better removal efficiency of 91.4% total nitrogen (T-N) and denitrification rate of 363.7 mg/L.day at a HRT of 3 h compared to ADS. Further, there was no nitrite accumulation in the DAS effluent. The nitrite accumulation in ADS effluent was lesser when CO₂ was used as buffer rather than K₂HPO₄ and KH₂PO₄. Estimation of kinetic parameters of hydrogenotrophic bacteria indicated lesser sludge production compared to heterotrophic denitrification. In the DAS, membrane fouling was nonexistent in the aeration reactor that was used to produce the recirculating effluent. On the contrary, membrane fouling was observed in the denitrification reactor that supplied hydrogen to the mixed liquor. Thus, this study demonstrated DAS capability in maintaining the acceptable water quality appropriate for aquaculture, in which a closed recirculating system is typically used.