Optimization of cultivation conditions for enhancing biomass,polysaccharide and protein yields of Chlorella sorokiniana by response surface methodology

On the basis of multiple single‐factor studies, the best conditions for each factor were selected by comparison. Taking the biomass, polysaccharide and protein yields of chlorella as response values, the four‐factor and three‐level response surface design experiment was performed on four parameters that significantly affected the response values to further determine the optimal conditions. The optimal parameters of the culture process were nitrogen source (urea), carbon source (sodium carbonate), nitrogen concentration (15.80 mmol/L), carbon concentration (0.36 mmol/L), phosphorus concentration (0.2 mmol/L), magnesium concentration (0.33 mmol/L), calcium concentration (0.25 mmol/L), iron concentration (0.02 mmol/L), initial pH (7.0), inoculation amount (6%) and ventilation capacity (40 L/hr). According to this process, the biomass, polysaccharide and protein yield of Chlorella sorokiniana obtained from indoor culture were close to the predicted value, which were more than three times as much as before, reaching 3.58, 0.52 and 0.46 g/L respectively. Chlorella sorokiniana, which was cultured outdoor according to the optimized process, increased its biomass, polysaccharide and protein yield by more than 1.5 times. Chlorella sorokiniana was significantly different before and after optimized culture. This provides a good source for the research of polysaccharide and protein of C. sorokiniana and helps the further study of its structure and biological activity, which makes the further development of its function more possible.     

Phenotypic characterization and cultivation conditions of inhibitor-producing fungus isolated from marine sediment

ABSTRACT:   Tyrosinase inhibitor-producing fungus of Trichoderma sp. strain H1-7, which was isolated from marine sediment, was investigated by phylogenetic analysis and physiological characteristics. Phylogenetic analyses of the strain were similar to Trichoderma atroviride or T. viride . Physiological characteristics of the strain were similar to T. viride , and based on these results, it was identified as T. viride . Characterization of tyrosinase inhibitory activity of a culture supernatant of the strain was investigated. The inhibitory activity of the supernatant of the strain decreased after cultivation for more than 3 days. Furthermore, sea water was not essential for the production of the tyrosinase inhibitor (TI). When TI production of the strain was compared to T. viride , the strain showed higher activity than T. viride . From this result, it seemed that the strain had characteristic features comparable to T. viride , which was isolated from the terrestrial environment. As TI production of the strain showed higher potential than that of T. viride , it is necessary to elucidate the chemical structure of TI exemplified in the present study.     

Biological evaluation of phytoplankton (Chlorella sp., Tetraselmis sp. and Isochrysis galbana) as food for milkfish (Chanos chanos) fry

Phytoplankton cultures of Chlorella sp., Tetraselmis sp. and Isochrysis galbana were used alone as feed to rear separate batches of newly caught milkfish (Chanos chanos) fry (about 21 days old). Ultrastructural studies of fry hepatocytes and pancreatic acinar cells indicated that they cannot directly utilize Chlorella, which has a rigid cell wall. The fry can directly utilize Tetraselmis and Isochrysis, but neither is nutritionally adequate for growth and survival if used as the only feed. Tetraselmis is nutritionally inferior to Isochrysis.     

Submersion time, depth, substrate type and sampling method as variation sources of marine periphyton

Periphyton is an additional food source in African and Asian brackish and freshwater fish ponds. The present study was a preliminary assessment of periphyton development on artificial substrates in temperate marine ponds. The effects of submersion time, substrate type, water depth, and total or partial sampling methods on the quantity and quality of periphyton collected, were evaluated. Four types of substrate (W: wooden poles, S: smooth fiber-glass strips, m: mosquito screen (1 mm-mesh) and M: garden netting (5 mm-mesh)) were deployed in a marine pond, and periphyton was collected 15 and 30 days later. The total amount of periphyton per substrate unit was collected as one sample or as 5 sub-samples. Results showed that (i) periphyton biomass in a marine pond increased between day 15 and day 30, (ii) more periphyton was collected on mosquito screen than on wooden poles, fiberglass strips and garden netting, (iii) periphyton biomass increased with submersion depth, (iv) sub-sampling leads to an underestimate compared to whole unit sampling, and (v) a correction of periphyton weight must be carried out considering the dissolved inorganic salts present in periphyton samples from marine and brackish ponds. Whole substrate unit sampling using a tube and stopper is recommended to avoid underestimation of periphyton development. Finally, the autotrophic fraction in the periphyton communities was very low compared to periphyton developed on biodegradable substrates in fertilized tropical ponds. Studies on fertilization and use of biodegraded substrates (i.e. long-time submerged wood) are recommended to further optimize periphyton development in temperate marine ponds.     

太空搭载小球藻（Chlorella vulgaris）子代群体新品系的筛选、培养及其营养组成分析

本文以经过太空搭载返回地面后保存的小球藻（Chlorella vulgaris）藻种T0为实验材料,从提高小球藻生长速度和促进小球藻油脂富集两个方面考虑,对小球藻生长的环境因子和培养基成分进行探讨和研究;最终经过两次筛选获得速长-油脂高产的株系TY31.实验结果表明,小球藻在140 μmol·m-2·s-1光照强度、23℃培养温度、以3.75 mmol/L的NH2 CONH2为氮源、以0.12 mmol/L的K2HPO4为磷源的最佳培养条件下,细胞生长速度加快,生长期变长,蛋白、脂肪及脂肪酸含量增加.研究发现,以第一次筛选的小球藻藻株T9为出发株,采用改良的BG-11培养基进一步选育的速长-油脂高产株系TY31,其最终细胞密度增加11.06×106 mL-1,脂肪含量增加6.50％,脂肪酸含量增加3.76％,具有应用于生物能源研究的潜力,为海洋微藻的开发利用奠定了基础.     

Evaluation of floating cages as an experimental tool for marine shrimp culture studies under practical earthen pond conditions

The New Caledonia blue shrimp Litopenaeus stylirostris is commercially produced under semi-intensive rearing conditions. The size of the farming earthen ponds (5–10 ha) and the subsequent production constraints make it difficult to use them as experimental units for random experiments. Moreover, since every pond has its own characteristics, ponds' effects cannot be ruled out, thus making it hard to define true replicates.In order to design future experiments under conditions resembling those used in production, we evaluated the possible use of floating cages as experimental units with the aim of assessing treatment effects with a reasonable statistical power. To this end, two sets of floating cages were placed respectively in two different ponds in a commercial farm. In both cases the zootechnical conditions in the cages were similar in post-larvae origin, management, and diet and feeding regime.The aim of the study was to evaluate on a technical basis the possibility of rearing shrimps in floating cages set up in earthen ponds and to assess this method from a statistical standpoint. Shrimps reared in and outside the cages showed comparable growth and survival rates. The variability in the zootechnical parameters between cages, expressed as the estimated coefficient of variation (CV) for survival rate, total final biomass (g), final body weight (g), daily increment in body weight (g day^− 1) and FCR were 11.0, 13.7, 4.4, 5.4, and 7.0%, respectively. On the basis of these figures, we calculated for a given statistical power (80%) the number of replicates (cages) that would be required to reveal significant differences between two treatments, at a 5% level of significance. We found that for expected differences of 20% from the control mean, 3 and 6 floating cages per treatment would be reasonable to determine statistical differences for growth parameters and survival rate, respectively. Moreover, we showed a significant pond effect in regard to survival and growth between the two sets of cages. These results illustrated the within-farm variability among the ponds, and confirmed that the specific characteristics of each pond from the same farm make it difficult to use the ponds themselves as experimental units.The study demonstrates that rearing in floating cages is an economical, powerful and sensitive experimental tool for shrimp culture studies specifically carried out under conditions close to semi-intensive production.     

固定化菱形藻（Nitzschia sp.）的生长及营养成分变化

选用底栖硅藻菱形藻(Nitzschiasp.)为实验对象,分别研究了CaCl2浓度、胶珠直径、接种密度及胶珠密度对其生长的影响及后两因素对其主要营养成分含量变化的影响。结果表明:(1)随培养时间的延长,CaCl2浓度对菱形藻生长有极显著影响,在2%CaCl2溶液中形成的胶珠里菱形藻比生长速率最高;(2)胶珠直径对菱形藻在初期和指数期的生长影响显著,且较小直径(2.5～3.0mm)的胶珠较适宜菱形藻生长;(3)随接种密度和胶珠密度的提高,菱形藻的比生长速率都有先增高后下降的变化趋势,接种密度较高(2.50×104～5.50×104mL-1)时,固定化组的比生长速率是对照组的2倍左右。胶珠密度为220粒/(100mL培养液)左右较适宜(胶珠直径3.0mm);(4)初始接种密度和胶珠密度试验中,蛋白质、总糖及粗脂肪的含量固定化组低于或相当于对照组,而叶绿素的含量固定化组高于对照组。     

小球藻和盐藻利用石油烃富集油脂的研究

利用单细胞分离和紫外诱变技术分别获得海水小球藻和盐生杜氏藻的生长优势株,将其分别接种于不同石油浓度的海水培养液中,利用紫外和荧光分光光度法分别测定培养液中带共轭双键的烃类化合物和芳烃的含量,利用索氏提取法测定胞内油脂含量。结果表明,石油浓度为1.5~10.0μg/ml的培养液中,小球藻和盐藻均能有效降解带共轭双键的烃类化合物,降解率分别为25.3%~35.5%和17.9%~24.0%,芳烃降解率分别为22.1%~30.2%和18.7%~26.2%;石油浓度为3.5μg/ml时,两种微藻对带共轭双键的烃类化合物和芳烃的降解效率均最大,小球藻的降解能力略好于盐藻;石油浓度为1.5μg/ml和3.5μg/ml时,两种微藻的胞内油脂含量分别占细胞干重的14.0%和25.5%,分别是对照组的1.5倍和1.2倍,浓度为3.5μg/ml时两种微藻的胞内油脂含量均最高。该研究为利用石油污染海水培养微藻使其富集油脂及今后开发微藻燃料奠定了基础。     

Enrichment of the nematode Panagrolaimus sp., a potential live food for marine aquaculture,with essential n-3 fatty acids

The nematode Panagrolaimus sp. (strain NFS-24-5) may have potential as a live food organism for larvae of several marine species. It can be mass-produced in liquid culture and is desiccation tolerant enabling long-term storage and transportation. Nematodes lack the essential fatty acid docosahexaenoic acid (DHA), so this investigation examined an enrichment procedure by incubating nematodes in S.presso^® (INVE Aquaculture, Belgium), a commercial enrichment product. Mass-produced nematodes from liquid cultures on yeast cells were cleaned and then exposed to concentrations of 0.1–3 % S.presso^® for 24 h at 200,000 nematodes ml^?1. Nematode viability was >90 % after the treatment. Following enrichment percentage, total lipid ranged from 23.6 to 33.3 % of nematode dry matter, and nematodes incubated in 3 % S.presso^® had a significantly higher lipid percentage than untreated controls. Enrichment was successful in increasing the percentage of DHA, and a maximum value of 5.8 % of total fatty acids was achieved. The results of enrichment of Panagrolaimus sp. (strain NFS-24-5) following treatment with 3 % S.presso^® should promote the carrying out of feeding trials to test the efficacy of the nematodes as a live food for larval marine fish and crustaceans.     

Mortality of scallop spat in cultivation, infested with tube dwellingbristle worms, Polydora sp.

Between 1995 and 1997, a traditional Norwegian oyster poll with an attachednursery was used for the production of scallop (Pecten maximus) spat.During summer 1997, four batches of 2 mm scallop spat were placed in thenursery. In June, hydroids (Obelia) caused problems with fouling andaccumulation of sediment in the nursery. Thereafter, the scallop spat wereheavily infested with tube dwelling polychaets (Polydora sp.). Thepolychaets were both introduced with poll water and reproduced inside thenursery. Infested spat suffered high mortalities. Although the spat could have been affected by the hydroids, as well as shortage of food, Polydorainfestations were considered the main cause of the mortalities. Dead andinfested spat were continuously sorted out and discarded, but the infestationproblems persisted. As a result, the scallop spat did not represent anycommercial value for the company, and in September, the nursery wasemptied. In total, approximately one million spat – representing onethird of Norway's intensive scallop spat production in 1997, was lost.     

磷限制及恢复对小球藻叶绿素荧光特性的影响

研究了小球藻（Chlorella sp．）一次性培养过程中,培养基中不同磷浓度（0、10、36．3和290．4μM）对其叶绿素荧光参数、细胞密度和叶绿素含量的影响以及磷添加后各荧光参数的瞬时变化（nutrient induced fluorescnce transients,NIFY反应）及恢复情况。培养温度为20±1℃,盐度为31,光照强度为100μmol·m^-1·s^-1。单因子方差分析的结果表明,随着培养时间的延长,磷浓度对小球藻的光合作用及生长均有显著影响（P〈0．05）。多重分析的结果表明,小球藻的最适磷浓度为36．3μM。小球藻的最大光能转化效率（Fv/／Fm）、实际光能转化效率（ФPSII）、电子传递效率（ETR）、光化学淬灭（qP）、细胞密度以及叶绿素含量随着起始磷浓度的增大而增大,在磷浓度为36．3μM达到最大值。磷重新添加后,瞬时荧光不断下降,ФPSII出现瞬间的上升后恢复至原初状态,qP、非光化学淬灭（NPQ）则不断上升。限制7d后重新添加磷,各处理组的Fv／Fm、ФPSII、ETR、NPQ 24h内基本恢复,而对照组的变化不显著。     

营养方式对小球藻生长性能及营养价值的影响

分别对小球藻Chlorellasp.2003ZGH023进行光合自养、混合营养高碳、混合营养低碳和异养低碳培养,探讨不同营养方式对小球藻生长及细胞营养组成的影响。结果表明,在基础培养基中添加有机碳源,可显著提高小球藻的细胞密度和生物量,其中异养低碳组细胞密度可达54.73×106cell/ml,生物量6.86 g(dw)/L,且混合营养和异养小球藻细胞体积变大。在细胞营养组成上,与自养小球藻细胞相比,异养培养的小球藻细胞内蛋白含量显著下降而脂肪含量显著增加。光合自养组、混合营养高碳组、混合营养低碳组和异养组小球藻细胞粗蛋白含量分别是41.88%±0.14%、24.60%±0.07%、21.93%±0.13%和12.91%±0.35%。不同营养方式下小球藻的氨基酸总量变化与其粗蛋白含量的变化趋势相一致,光合自养组>混合营养高碳组>混合营养低碳组>异养组。小球藻总脂肪含量最高的为混合营养高碳组(15.32%±1.58%)和异养低碳组(14.15%±0.93%),显著高于混合营养低碳组(12.35%±0.25%)和光合自养组(10.04%±0.39%)。小球藻的脂肪酸组成会随着营养方式的改变而改变,光合自养组小球藻...     

寄生海水养殖鱼类的拟格拉夫涡虫的流行、危害与防治

葡萄糖激酶（GK；E．-C．2．7．1．1）是己糖激酶家族成员，葡萄糖激酶在控制葡萄糖利用率保持葡萄糖平衡方面起着重要的作用。但是，鱼类对糖的利用率却较低，目前对此较公认的解释是由于鱼类肝脏缺乏可诱导的GK活力而不能有效地使细胞内的葡萄糖转化为6-磷酸葡萄糖，可这一解释和现有有关鱼类存在有功能的GK样酶活力以及饲料中碳水化合物对GK表达的诱导作用明显有矛盾。本文报导了从翘嘴红鲐肝脏中分离到的GKcDNA全序列，对其序列进行了比较和分析，为进一步研究翘嘴红鲐GK酶在不同营养条件下的表达奠定基础。     

寄生海水养殖鱼类的拟格拉夫涡虫的流行、危害与防治

自2000年10月中旬,在罗源湾发现一种罕见的能够寄生鱼类的涡虫,对其流行病学、形态学以及进化地位进行了大量的研究,结果表明：这种涡虫是一种未被报道过的新种,在福建省的各海水养殖区广泛流行,能够寄生于眼斑拟石首鱼（Sciaenops ocellatus）、大黄鱼（Pseudosciaena crocea）、鮸鱼（Miichthys miiuy）、牙鲆（Paralichthys olivaceus）、黑鲷（Sparus macrocephalus）和横纹东方鲍（Takifugu oblongus）等海水养殖鱼类的鳃、鳍及体表,造成寄生部位的严重损伤,鱼因呼吸困难或细菌的继发性感染而造成大量死亡,死亡率可达20％-60％;流行季节主要为秋季（8-11月）;水温22-26℃,盐度25-30。此外,提出了使用地下水,降低盐度、温度,合理用药等切实可行的防治本涡虫病的建议。     

响应面法优化芽孢杆菌(Bacillus sp.)A4的培养参数

为了提高芽孢杆菌(Bacillus sp.)A4的菌浓度,采用响应面法对其培养参数进行优化。以单因素实验为基础,筛选适合A4菌生长的碳源和氮源,采用Plackett-Burman实验方法确定影响菌浓度的显著因子;通过最陡爬坡实验逼近菌浓度最大值响应稳定区域;最后根据Box-Behnken实验确定显著因子的最佳水平,建立相关参数的回归方程,得到优化培养参数,并以摇瓶培养实验验证理论参数及方程的科学性。结果显示,最适碳源、氮源分别为玉米浆、大豆蛋白,且它们与温度均为显著影响A4菌浓度的因子,其最适水平分别为20.06 g·L~(-1)、13.29 g·L~(-1)、30.26℃。采用优化后的参数进行摇瓶培养(24 h),A4菌浓度达1.19×109CFU·mL~(-1),与理论计算值(1.24×109CFU·mL~(-1))无显著差异(P0.05),但显著高于未优化菌株培养浓度(2.69×107CFU·mL~(-1))(P0.01)。可见,运用响应面法优化芽孢杆菌A4的培养参数,能显著提高A4菌的菌浓度。     

Stable isotope analysis of trophic position and terrestrial vs. marine carbon sources for juvenile Pacific salmonids in nearshore marine habitats

Abstract  Stable isotope analysis was used to determine trophic position and the relative contributions of terrestrial-derived carbon (TDC) and marine-derived carbon for Chinook, Oncorhynchus tshawytscha (Walbaum), pink, Oncorhynchus gorbuscha (Walbaum), and chum, Oncorhynchus keta (Walbaum) salmon fry in near-shore marine habitats. Chum fry were enriched in δ ¹³C relative to pink fry, and enriched in δ ¹⁵N relative to both Chinook and pink fry. Between 5.5 and 39.7% of the carbon in the three species was TDC. The TDC was higher in chum fry (28.7 ± 4% SD) than in pink fry (24.9 ± 4.4% SD), but TDC did not differ between Chinook fry (27.8 ± 9.5% SD) and either chum or pink fry. The fry of these three species of Pacific salmon may form a trophic hierarchy with chum fry occupying the highest trophic position and the three species may also partition resources according to carbon source.     

Evaluation of the robustness of optical density as a tool for estimation of biomass in microalgal cultivation: The effects of growth conditions and physiological state

We evaluate the robustness of optical density (OD) as a tool in the assessment of the biomass of the cryptophyte Rhodomonas salina under different growth conditions. We measure the OD under three different wavelengths, 550, 665 and 750 nm. We find, as expected, that growth rates of the microalga depend strongly on growth conditions, being highest in saturating light conditions under non‐limiting nitrogen (N) availability. OD – cell count relationships are strong and well defined regardless of the growth conditions and wavelength used for measurement. However, measuring within the absorption range of chlorophyll, at 550 and 665 nm, does give higher regression coefficients under conditions leading to a high cell chlorophyll content, while the coefficients of determinations are slightly higher when cell chlorophyll content is low. We conclude that to use OD as a proxy for biomass under large‐scale production of microalgae, it is important to take into account that the precise relationship between OD and algal biomass does depend on factors such as irradiance and nutrient availability, and hence the physiological state of the microalgae as well as the production conditions. Errors in estimation of biomass may range from 44% to 95%, if these factors are not taken into consideration.     

Influence of food concentration and abiotic parameters on population density growth of the ciliated marine protozoan Euplotes sp. under controlled conditions

High mortality is common when culturing most marine fish larvae, especially during the transition from endogenous to exogenous feeding. In aquaculture, many species of marine fish are not able to survive when only fed enriched rotifers and Artemia spp. nauplii. Ciliates are a potential alternative live food organism for first‐feeding larvae, because they can grow to high population densities, accept inert diets, and are natural prey organisms of marine fish larvae. The range of culture parameters to optimize population growth of the ciliate Euplotes sp. are unknown. Five experiments were conducted to determine the effects of food concentration and abiotic factors including salinity, aeration rate, temperature and photoperiod on population growth of the ciliate Euplotes sp. Results indicated the optimal ranges for population growth of Euplotes sp. was a temperature between 26 and 32°C, salinities from 20 and 35 g/L, food (Protein Selco®) concentrations of 250 and 500 mg/million ciliates, absence of or low aeration (8.5 cm³/min) and the photoperiod 0L:24D. Euplotes sp. can tolerate high ammonia and very low dissolved oxygen concentrations, and population growth can occur in these conditions for at least 7 days.     

Evaluation of Spent Bleaching Clay from Palm Oil Refining as an Ingredient for Diets of Red Hybrid Tilapia,Oreochromis sp.

Abstract

A feeding trial was conducted to determine whether spent bleaching clay (SBC) from palm oil refining could be used as a diet ingredient in the diets of red hybrid tilapia, Oreochromis sp. Five practical diets containing 0, 10, 20, 30, or 40% SBC were formulated and fed to triplicate groups of hybrid tilapia fingerlings (mean initial weight of 3.9±0.1 g) for 8 weeks. All diets were formulated to be isonitrogenous (31% crude protein) and isolipidic (13% crude lipid). There was a trend of improved pellet stability in water as the level of SBC in the diet was increased. Growth and feed utilization efficiency of hybrid tilapia fed up to 40% SBC was mostly not significantly different (P > 0.05) compared to fish fed the control diet without any SBC. Survival, bone ash, hepatosomatic index and hematocrits of hybrid tilapia were also not affected by dietary treatments. Whole-body lipid content increased and body moisture decreased significantly with increasing dietary SBC. It was concluded that SBC from palm oil refining is a potential diet additive in tilapia diets offering a cheap source of dietary lipid (and energy), imparting pellet stability in water and possibly acting as a mycotoxin absorber.