Optimization of fermentation conditions for 1,3-propanediol production by marine Klebsiella pneumonia HSL4 using response surface methodology

The industrially important organic compound 1,3-propanediol(1,3-PDO) is mainly used as a building block for the production of various polymers. In the present study, response surface methodology protocol was followed to determine and optimize fermentation conditions for the maximum production of 1,3-PDO using marine-derived Klebsiella pneumoniae HSL4. Four nutritional supplements together with three independent culture conditions were optimized as follows: 29.3 g/L glycerol, 8.0 g/L K 2 HPO 4, 7.6 g/L(NH 4) 2 SO 4, 3.0 g/L KH 2 PO 4, pH 7.1, cultivation at 35°C for 12 h. Under the optimal conditions, a maximum 1,3-PDO concentration of 14.5 g/L, a productivity of 1.21 g/(L·h) and a conversion of glycerol of 0.49 g/g were obtained. In comparison with the control conditions, fermentation under the optimized conditions achieved an increase of 38.8% in 1,3-PDO concentration, 39.0% in productivity and 25.7% in glycerol conversion in flask. This enhancement trend was further confirmed when the fermentation was conducted in a 5-L fermentor. The optimized fermentation conditions could be an important basis for developing lowcost, large-scale methods for industrial production of 1,3-PDO in the future.     

Effect of nitrate and phosphate on accumulation of β-carotene isomers inDunaliella salina

Dunaliella salina, a halotolerant unicellular green alga, can accumulate a large amount of β-carotene under certain environmental conditions. The isomers of β-carotene extracted fromD. salina cultured in medium with different nitrate and phosphate concentrations were analysed by HPLC with Alox-T alumina column. At least six isomers were found in different proportions depending on the culture media's nitrate and/or phosphate concentrations. Nitrate and/or phosphate deficiency was conducive to the accumulation of total cis isomers but not of all trans isomer. It is suggested that 1 mmol/L KNO₃ and 0.1 mmol/L KH₂ PO₄ are favourable for accumulation of total cis β-carotene. Contribution No. 2090 from the Institute of Oceanology, Chinese Academy of Sciences. This project was supported by the National Natural Science Foundation of China, Grant No. 38970587.     

Studies on culture conditions of benthic diatoms for feeding abalone

Effects of salinity, pH, nitrogenous and phosphate nutrients on the growth rate of four species of benthic diatoms were studied by using the method of in situ optical density measurement. The optimal culture conditions for the four species of diatoms are as follows: forAmphora coffeaeformis, s 35; pH 8.5; NO₃ ⁻−N 1.54, NH₄ ⁺−N, 7.0; CO (NH₂)₂−N, 2.5 (mg/L); PO₄ ³⁻−P 1.13 (mg/L); forCocconeis scutellum varparva, s 30; pH 8.5; NO ₃ ⁻ −N 3.08, NH ₄ ⁺ −N 3.5, CO(NH₂)₂−N 5.0 (mg/L); pO ₄ ³⁻ −P 0.283 (mg/L); forNavicula corymbosa s 25; pH 8.0; NO ₃ ⁻ −N 1.54, NH ₄ ⁺ −N 3.5 CO(NH₂)₂−N 5.0 (mg/L); PO ₄ ³⁻ −P 0.565 (mg/L); forNavicula mollis. s 25; pH 8.0; NO ₃ ⁻ −N 1.54, NH ₄ ⁺ −N 1.75, CO(NH₂)₂−N 1.25 (mg/L); PO ₄ ³⁻ −P 0.141 (mg/L). Part of the results of this paper were exchanged in the Fourth Asian Aquaculture Forum. Oct. 16–20. 1995. Beijing, P. R. China.     

A new pullulan-producing yeast and medium optimization for its exopolysaccharide production

1　Introduction　Pullulanstandsforthewater solublehomopolysac charideproducedextracellularlybythe polymorphicmicromyceteAureobasidium pullulans (Yuenetal.,1974 ;Marketal.,1988) .Itisalinearα D glucan ,mademainlyofmaltotrioserepeatingunitsintercon nectedbyα 1,6 linkages.Theregularalternationofα 1,4andα 1,6bondsresultsintwodistinctiveprop ertiesofstructuralflexibilityandenhancedsolubility(Sanjeevetal.,2 0 0 1) .Thispolysaccharideisofeco nomicimportancewithincreasingapplicationinfood ,phar…     

Bioflocculant Produced by Klebsiella sp. MYC and Its Application in the li＇eatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.     

The eco-nutrition requirements for dietary protein and its rhomb characteristics in juvenile turbot (Scophthalmus maximus L.)

We evaluated the dietary protein requirements of juvenile turbot (Scophthalmus maximus L.) and their effects on aquatic quality. Five experimental diets were formulated containing 450, 480, 500, 520, and 540 g/kg. Each diet was randomly assigned to triplicate groups of juvenile turbot (mean initial body weight 34.5 ± 5.5 g) for 88 d. Both the weight gain ratio and feed efficiency increased with increasing dietary protein up to 500 g/kg, but no further improvement was detected when dietary protein levels were >500 g/kg. Protein intake and digestion increased with protein levels, while fecal nitrogen and nitrogen content in seawater increased only when dietary protein exceeded 500 g/kg. Protein digestibility was highest at intermediate dietary protein levels. Chemical oxygen demand, nitrite-nitrogen (NO2--N) and phosphatic-phosphor (PO43--P) levels increased in the rearing water as dietary protein levels increased. The optimum eco-nutrition level of dietary protein for juvenile turbot was 500 g/kg under the current experimental conditions. The diets containing 540 and 500 g/kg protein had similar growth rates and feed conversion ratios, but levels of ammonia (NH4+) and nitrogen were considerably higher in the water and feces, respectively, at the higher level of dietary protein. The difference in the pattern of change between body weight gain and ammonia concentration in water with increasing dietary protein is described by rhomb characteristics.     

Optimization of dilute acid hydrolysis of Enteromorpha

Acid hydrolysis is a simple and direct way to hydrolyze polysaccharides in biomass into fermentable sugars. To produce fermentable sugars effectively and economically for fuel ethanol, we have investigated the hydrolysis of Enteromorpha using acids that are typically used to hydrolyze biomass:H2SO4, HCl, H3PO4 and C4H4O4 (maleic acid). 5%(w/w) Enteromorpha biomass was treated for different times (30, 60, and 90 min) and with different acid concentrations (0.6, 1.0, 1.4, 1.8, and 2.2%, w/w) at 121°C. H2SO4 was the most effective acid in this experiment. We then analyzed the hydrolysis process in H2SO4 in detail using high performance liquid chromatography. At a sulfuric acid concentration of 1.8% and treatment time of 60 min, the yield of ethanol fermentable sugars (glucose and xylose) was high, (230.5 mg/g dry biomass, comprising 175.2 mg/g glucose and 55.3 mg/g xylose), with 48.6% of total reducing sugars being ethanol fermentable. Therefore, Enteromorpha could be a good candidate for production of fuel ethanol. In future work, the effects of temperature and biomass concentration on hydrolysis, and also the fermentation of the hydrolysates to ethanol fuel should be focused on.     

The inhibitory degree between Skeletonema costatum and dinoflagllate Prorocentrum donghaiense at different concentrations of phosphate and nitrate/phosphate ratios

Interactions between Skeletonema costatum (S. costatum) and Prorocentrum donghaiense (P. donghaiiense) were inves-tigated using bi-algal cultures at different concentrations of phosphate (PO4-P) and nitrate/phosphate (N/P) ratios. Experiments were conducted under P-limited conditions and the Lotka-Volterra mathematical model was used to simulate the growth of S. costatum and P. donghaiense in the bi-algal cultures. Both of these two species were inhibited significantly in bi-algal culture. The results of the simulation showed that the inhibitory degree of S. costaum by P. donghaiense was high when the concentration of PO4-P was low (0.1μmolL-1/2 d), but that of P. donghaiense by S. costaum was high with increased PO4-P supply (0.6μmolL-1/2 d). At low concen-tration of PO4-P (0.1μmolL-1/2 d), or high concentration of PO4-P (0.6μmolL-1/2 d) with high N/P ratio (160), the interactions be-tween S. costatum and P. donghaiense were dependent on the initial cell densities of both species. At high concentration of PO4-P (0.6μmolL-1/2 d) with low N/P ratio (25 or 80), S. costatum exhibited a survival strategy superior to that of P. donghaiense. The de-gree of inhibition of P. donghaiense by S. costaum increased with elevated N/P ratio when the medium was supplemented with con-centration 0.1μmolL-1/2 d of PO4-P. The degree of inhibition to P. donghaiense by S. costaum increased with elevated N/P ratio at low concentration of PO4-P (0.1 μmolL-1/2 d). This trend was conversed at high concentration of PO4-P (0.6μmolL-1/2 d). However, the degree of inhibition of S. costaum by P. donghaiense increased with the increased N/P ratio at different PO4-P concentrations (0.1μmolL-1/2 d and 0.6μmolL-1/2 d). These results suggested that both phosphate concentration and N/P ratio affected the competition between S. costaum and P. donghaiense: P. donghaiense is more competitive in environments with low phosphate or high N/P ratio and the influence of N/P ratio on the competition was more significant with lower phosphate concentration.     

Characteristics of nutrients in the Jiulong River and its impact on Xiamen Water, China

Water samples were collected at 20 sites on 4 occasions in 2009(twice in May,and once in both August and November) along the Jiulong River,South China to examine how nutrient inputs from the Jiulong River could affect the nutrient status of the Xiamen Water.Samples were analyzed for nitrite(NO2-N),nitrate(NO3-N),ammonium(NH4-N),phosphate(PO4-P),silicate(SiO3-Si),salinity,and temperature,to determine the nutrient and trophic status of the river.The results indicate that nutrients are derived mainly from river runoff.NO3-N was the main form of DIN in most parts of the river.In addition,NO3-N,DIN,and SiO3-Si behave conservatively.There is a surplus of DIN and SiO3-Si in the river,and PO4-P is a limitation on phytoplankton growth.The concentration of DIN is typically above 0.60 mg/dm3,and higher than 1.00 mg/dm3in most parts of the river.The concentration of PO4-P is typically above 0.02 mg/dm3,while the concentration of SiO3-Si is higher than 1.00 mg/dm3.Between 2003 and 2008,samples were collected 3 times per year(May,August and November) at 27 sites in the Xiamen Water and analyzed for NO2-N,NO3-N,NH4-N,PO4-P,salinity,and temperature.We discovered that the Jiulong River was the key source of DIN into the Xiamen Water,but not PO4-P,indicating the reason of the N/P molar ratio imbalance in the Xiamen Water.In the future,the effects of high DIN concentrations on the phytoplankton communities and marine ecosystems of the Xiamen Water shall be studied.     

Effect of nitrogen to phosphorus ratios on cell proliferation in marine micro algae

The ratio of nitrogen/phosphorus (N/P) is known to affect cell proliferation of some marine micro algae. We evaluated the effect of N/P ratios on the proliferation and succession of phytoplankton using five marine micro algae species. We used two sources of nitrogen, NH₄Cl (N₁) and urea (N₂), and a single source of phosphorous, NaH₂PO₄(P). The optimal N/P ratio that differed among the five species was affected by the source of nitrogen, being as follows (N₁/P, N₂/P in order): Thalassiosira sp. (30/1, 20/1), Heterosigma akashiwo (30/1, 30/1), Chroomonas salina (20/1, 30/1), Chaetoceros gracilis (40/1, 60/1), and Alexandrium sp. (10/1, 30/1). Thus, the source of nitrogen must be considered when analyzing the N/P ratio. Our results provide insight for predicting phytoplankton succession in coastal waters and may be used to forecast the potential risk of harmful algal blooms.     

A STUDY ON THE RELATIONSHIPS OF THE NUTRIENTS NEAR THE CHANGJIANG RIVER ESTUARY WITH THE FLOW OF THE CHANGJIANG RIVER WATER

Investigations from August, 1985 to July , 1986 showed that the high concentration area of PO4-P , SiO3-Si and NO3-N gradually reduced with the reduction of the area of the Changjiang River diluted water from summer, autumn to winter , and that the seasonal distributions and variations of the nutrients concentrations were mainly controlled by the river flow and were also related to the growth and decline of phytoplankton . The conservation of SiO3-Si and NO3-N in the estuary in the flood season was poorer than that in the dry season .. The behaviour of PO4-P in the estuary shows that aside from -biological removal, buffering of PCU-P is possible in the estuary . The highest monthly average concentrations and annual average concentrations in the river mouth were respectively 0.88 and 0.57 umol/L for PO4-P,191.5 and 96.2 umol/L for SiO3-Si, and 81.6 and 58.6 umol/L for NOs-N . The Changjiang's annual transports of PO4-P , SiO3-Si and NO3-N to the sea were about 1.4×104tons , 204.4×104 tons and 63.6×104     

Nutrient and eutrophication characteristics of the Dongshan Bay,South China

We recorded NO 3-N, NO 2-N, NH 4-N, PO 4-P, SiO 3-Si, salinity, and temperature data at 10 stations in the Dongshan Bay in May, June, July, August, October, and November 2008, analyzing nutrient and eutrophication characteristics. The mean concentration of dissolved inorganic nitrogen(DIN) was 0.30–0.40 mg/dm 3; generally, NO 3-N was the main form in most areas. The mean concentrations of PO 4-P and SiO 3-Si were 0.040–0.060 mg/dm 3 and 1.00–1.50 mg/dm 3, respectively. We proved that the majority of the SiO 3-Si in the Dongshan Bay came from the Zhangjiang River, with some coming from the Bachimen Strait. DIN originated from both the Zhangjiang River and the Bachimen Strait. Most PO 4-P originated from the Bachimen Strait, and some came from the Zhangjiang River. We found that P was an overall limiting factor to the phytoplankton community in most of the Dongshan Bay, and that Si and N were in surplus. However, near the Bachimen Strait Si became a limiting factor, especially for diatoms, while P and N were in comparative surplus. We used a potential eutrophication assessment method to analyze eutrophication, and showed that the most serious eutrophication occurred near the Zhangjiang River estuary and near the Bachimen Strait. In 2008, DIN levels were four times higher than that in 1988; PO 4-P levels were threefold higher, while SiO 3-Si was approximately double. Dissolved nutrients increased between 1988 and 2008. DIN increased at the greatest rate comparing to PO 4-P and SiO 3-Si, thus the N/P and N/Si mol ratios increased. Further studies on the effects of high DIN concentrations on the phytoplankton communities and marine ecosystems of the Dongshan Bay are needed.     

Study on gametophyte vegetative growth ofUndaria pinnatifida and its applications

When cultured under certain environmental conditions (25°C, light intensity 80 μmol/m²·s, LD 12/12, in enriched seawater medium with 7×10⁻⁴ mol/L NO₃−N, 1.56×10⁻⁴ mol/L, PO₄−P and supplements of other elements like Mn, Fe, I, etc.), male and female gametophytes ofU. pinnatifida kept growing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. The empirical formulaG _m=G _o·3^m was established to estimate the output of vegetative gametophytes. Vigorous vegetative gametophyte cells began to form reproductive structures (oogonium and spermatangium, when the temperature was lower than 25°C and other environmental factors were kept optimal. The sufficient supply of gametophyte cells provided enough seeds for raisingUndaria sporelings on production scale. Controlled cross-breeding experiments using selected male and female gametophyte clones which increase their cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes from the cross-breeding had almost the same length and width increase rates as those of the control. The fact that vegetative gametophytes can be purposely selected, propagated quickly, cross-bred, with the resulting sporophytes having almost the same characteristics leads to a new way to select desiredUndaria strains for long time use without losing the desired economic characteristics. Contribution No. 2696 from the Institute of Oceanology, Chinese Academy of Sciences. Supported by National Natural Science Foundation of China.     

Optimization of Shanghai Marine Environmental Monitoring Sites in the Identification of Boundaries of Different Water Quality Grades

Water quality is critical to ensure that marine resources and the environment are utilized in a sustainable manner. The objective of this study is therefore to investigate the optimum placement of marine environmental monitoring sites to monitor water quality in Shanghai, China. To improve the mapping or estimation accuracy of the areas with different water quality grades, the monitoring sites were fixed in transition bands between areas of different grades rather than in other positions. Following bidirectional optimization method, first, 18 candidate sites were selected by filtering out specific site categories. Second, three of these were, in turn, eliminated because of the rule defined by the changes in the areas of water quality grades and by the standard deviation of the interpolation errors of dissolved inorganic nitrogen (DIN) and phosphate (PO₄-P). Furthermore, indicator kriging was employed to depict the transition bands between different water quality grades whenever new sampling sites were added. The four optimization projects of the newly added sites reveal that, all optimized sites were distributed in the transition bands of different water grades, and at the same time in the areas where the historical sites were sparsely distributed. New sites were also found in the overlap region of different transition bands. Additional sites were especially required in these regions to discriminate the boundaries of different water quality grades. Using the bidirectional optimization method of the monitoring sites, the boundaries of different water quality grades could be determined with a higher precision. As a result, the interpolation errors of DIN and PO₄-P could theoretically decrease.     

溶藻弧菌dtd基因的克隆及原核表达条件优化

根据NCBI数据库中已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列合成一对特异性引物,应用聚合酶链式反应(PCR技术)扩增溶藻弧菌HY9001菌株dtd基因,将其定向克隆到原核表达载体pET-32a构建重组表达质粒pET-DTD,并对其进行诱导温度、诱导时间、诱导IPTG浓度等条件的优化,最后探究其纯化时最佳咪唑洗脱浓度。结果表明:DTD蛋白成功表达,且其以包涵体的形式存在,在诱导温度37℃、IPTG浓度0.1 mmol/L条件下诱导5 h表达量最高,纯化最佳咪唑洗脱浓度为150 mmol/L。     

NUTRIENTS NEAR THE ARCTIC POLE REGION

The distributions of SiO_3-Si, PO_4-P, NO_3-N and NO_2-N concentrations in the seawater, iceand snow from 82°45′21″W, 88°01′20″N to the North Pole are reported and the exchange of thenutrients among the seawater, ice and snow are discussed. The average concentrations of the nutrients inthe seawater near the North Pole were 4.9±2.4μmol/L for SiO_3-Si; 0.60±0.10μmol/L for PO_4-P,3.4±1.7μmol/L for NO_3-N and 0.13±0.04μmol/L for NO_2-N. The nutrient concentrations inthe ice and snow in the North Pole were 0.20 and 0.65μmol/L for SiO_3-Si; 0.22 and 0.25μmol/L forPO_4-P; 1.6 and 5.8μmol/L for NO_3-N, 0.19 and 0.15μmol/L for NO_2-N.     

Evaluation of the retrieval of total suspended matter concentration in Taihu Lake,China from CBERS-02B CCD

Remote sensing techniques is used to quantify the total suspended matter concentration (C _TSM). In this study, we used remotely sensed data to retrieve the C _TSM for the Taihu Lake, China, and developed an atmospheric correction algorithm especially for CBERS-02B CCD data. We simulated the remote sensing reflectance (R _rs) of CCD bands using in-situ observations made in a cruise over the Taihu Lake in autumn 2004, from which a retrieval model is established with simulated R _rs(830) and measured C _TSM. In addition, we applied the atmospheric correction algorithm and retrieval model to process the CCD data over the lake in 2008 and to retrieve the C _TSM. The RMS relative error between the C _TSM retrieved from MODIS and from the CCD images is about 42.9%, indicating that algorithms described in this paper can be used for the application of CCD data in monitoring the C _TSM distribution in the Taihu Lake.     

Promotive effect of se on the growth and antioxidation of a blue-green algaSpirulina maxima

Cultures of a blue-green algaSpirulina maxima (Setch. et Gard.) Geitler with various concentrations of Se in Zarrouk's medium showed that not higher than 40 mg/L Se could promote its growth. The present experiments showed thatS. maxima grown under normal conditions, has an oxidant stress defence system for hydrogen peroxide (H₂O₂) removal, which is the Halliwell-Asada pathway. When 4 to 20 mg/L Se was added to the algal medium, this pathway was replaced by a so-called Sestressed pathway containing GSH peroxidase (GSH-POD). As a result of the occurrence of both higher activity of GSH-POD and lower levels of hydroxyl radical (OH·), the Se-stressed pathway scavenged H₂O₂ so effectively that the growth ofS. maxima was promoted by 4 to 20 mg/L Se. While GSH-POD activity of the alga disappeared at 40 mg/L Se, the recovery of ascorbate peroxidase was observed. The lower levels of ascorbic acid and GSH made the Halliwell-Asada pathway for scavenging H₂O₂ less effective, while the highest activity of catalase might be responsible in part for the H₂O₂ removal, causing the level of OH· inS. maxima grown at 40 mg/L Se to be much higher than the OH· level in this alga grown at 4 to 20 mg/L Se, but lower than that in the control. The OH· level changes caused the growth ofS. maxima cultured at 40 mg/L Se to increase slightly to close to that of the control.     

Optimization of culture conditions and medium composition for the marine algicidal bacterium Alteromonas sp. DH46 by uniform design

Harmful algal blooms (HABs) have led to extensive ecological and environmental issues and huge economic losses. Various HAB control techniques have been developed, and biological methods have been paid more attention. Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner, and kill or damage the algal cells. A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp. The culture conditions were optimized using a single-factor test method. Factors including carbon source, nitrogen source, temperature, initial pH value, rotational speed and salinity were studied. The results showed that the cultivation of the bacteria at 28°C and 180 r min^?1 with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46. The optimal medium composition for strain DH46 was determined by means of uniform design experimentation, and the most important components influencing the cell density were tryptone, yeast extract, soluble starch, NaNO₃ and MgSO₄. When the following culture medium was used (tryptone 14.0g, yeast extract 1.63g, soluble starch 5.0 g, NaNO₃ 1.6 g, MgSO₄ 2.3 g in 1L), the largest bacterial dry weight (7.36 g L^?1) was obtained, which was an enhancement of 107% compared to the initial medium; and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.     

Effects of nitrogen specification and culture method on growth of Enteromorpha prolifera

Eutrophication, which is the enrichment of a water mass with inorganic and organic nutrients that support plant growth, is a key factor in stimulating phytoplankton growth. In this study, we determined the effects of various nitrogen sources, different nitrogen concentrations in the culture medium, and two culture methods on the growth of the green alga, Enteromorpha prolifera. The relationship between the specific growth rate of E. prolifera and NO3--N concentration was consistent with that estimated using the Monod equation (R2 = 0.9713, P < 0.01). In the NO3--N medium, the maximum specific growth rate was calculated to be 0.1634/d and the semi-saturation constant was calculated to be 16.86 μmol/L. Our results show that E. prolifera can effectively utilize NH4+-N, NO3--N, and NO2--N and urea-N in the range of 5 to 50 μmol/L. NH4+-N was preferentially assimilated by E. prolifera, and urea-N was favorable for long-term growth.