Odontocete Stranding Patterns in the Main Hawaiian Islands 1937–2002): how do they compare with live animal surveys?

In this study we (1) synthesized 65 yr of odontocete stranding data around the main Hawaiian Islands (1937–2002); (2) analyzed stranding patterns and trends over time; and (3) compared occurrence patterns based on sightings of live animals with stranding data and evaluated the compatibility of these data sets. From 1937 to 2002, 202 odontocete strandings were recorded by the National Marine Fisheries Service, Pacific Islands Regional Office. Strandings increased through time due to increased reporting effort and occurred throughout the year. The four most common of 16 species reported were Kogia spp. (18%), spinner dolphins (Stenella longirostris) (15%), striped dolphins (Stenella coeruleoalba) (11%), and sperm whales (Physeter macrocephalus) (10%). The highest proportion of strandings was recorded on O‘ahu (48%), followed by Maui/La¯na‘i (24%), Kaua‘i (12%), Hawai‘i (11%), and Moloka‘i (5%). Comparison with four previously published live animal survey studies suggests that stranding records are a good indicator of species composition and yield reasonable data on the frequency of occurrence of species in the region they cover.

Abundance and distribution patters of Hawaiian odontocetes: focus on O'ahu

This dissertation is an assessment of the status of odontocetes in Hawaiian waters focussing on O´ahu. The work builds on available literature, and on data collected by the author and by others in Hawaiian waters. Abundance and distribution patterns of odontocetes were derived from stranding and aerial survey data. A stranding network operated by the National Marine Fisheries Service, Pacific Area Office collected 187 stranding reports throughout the main Hawaiian Islands between 1937 and 2002. These reports included 16 odontocete species. Number of stranding reports increased over time and was highest on O´ahu. Strandings occurred throughout the year. The difference in number of strandings per month was not significant. Fifteen of the 16 species reported in the stranding record for the main Hawaiian Islands were also reported by aerial survey studies of the area between 1993 and 1998. Only 7 of the species reported were detected during aerial transects around O′ahu between 1998 and 2000. Based on the stranding record, Kogia sp., melon-headed whales, striped dolphins and dwarf killer whale appear to be more common than suggested by aerial surveys. Conversely, pilot whales and bottlenose dolphins were more common, according to aerial surveys, than predicted by the stranding data. Aerial surveys of waters between 0 and 500m around the Island of O′ahu showed that the most abundant species by frequency of occurrence was the pilot whale (30% of sightings), followed by the spinner (16%) and bottlenose dolphin (14%). Because of small sample size, abundance estimates for odontocetes have a high level of uncertainty. The unavailability of a correction factor for g(0)<1, and the reduced visibility below the aircraft further reduced accuracy and increased the inherent underestimation in the data. The most abundant species according to distance sampling estimates were spotted dolphins, pilot whales, false killer whales and spinner dolphins. A natural factor shaping the ecology of odontocete populations is predation pressure both by other odontocetes and, more frequently, by sharks. An account of predation by a tiger shark on a spotted dolphin near Penguin Banks is used as an example of the potential mechanisms of predation by sharks on odontocetes.

Stocks of Dolphins (Stenella spp. and Delphinus delphis) in the Eastern Tropical Pacific: A Phylogeographic Classification

Current information is reviewed that provides clues to the intraspecific structure of dolphin species incidently killed in the yellowfin tuna purse-seine fishery of the eastern tropical Pacific (ETP). Current law requires that management efforts are focused on the intraspecific level, attempting to preserve local and presumably locally adapted populations. Four species are reviewed: pantropical spotted, Stenella attenuata; spinner, S. longirostTis; striped, S. coeruleoalba; and common, Delphinus delphis, dolphins. For each species, distributional, demographic, phenotypic, and genotypic data are summarized, and the putative stocks are categorized based on four hierarchal phylogeographic criteria relative to their probability of being evolutionarily significant units. For spotted dolphins, the morphological similarity of animals from the south and the west argues that stock designations (and boundaries) be changed from the current northern offshore and southern offshore to northeastern offshore and a combined western and southern offshore. For the striped dolphin, we find little reason to continue the present division into geographical stocks. For common dolphins, we reiterate an earlier recommendation that the long-beaked form (Baja neritic) and the northern short-beaked form be managed separately; recent morphological and genetic work provides evidence that they are probably separate species. Finally, we note that the stock structure of ETP spinner dolphins is complex, with the whitebelly form exhibiting characteristics of a hybrid swarm between the eastern and pantropical subspecies. There is little morphological basis at present for division of the whitebelly spinner dolphin into northern and southern stocks. However, we recommend continued separate management of the pooled whitebelly forms, despite their hybrid/intergrade status. Steps should be taken to ensure that management practices do not reduce the abundance of eastern relative to whitebelly spinner dolphins. To do so may lead to increased invasion of the eastern's stock range and possible replacement of the eastern spinner dolphin genome.(PDF file contains 24 pages.)

Review of geographical stocks of tropical dolphins (Stenella spp. and Delphinus delphis) in the eastern Pacific

Information on geographical variation is reviewed for Stenella attenuata, S. longirostris, S. coeruleoalba, and Delphinus delphis in the eastern tropical Pacific, and boundaries for potential management units are proposed. National Marine Fisheries Service and Inter-American Tropical Tuna Commission sighting records made from 1979 to 1983 which were outside boundaries used in a 1979 assessment were examined for validity. Tagging returns and morphological data were also analyzed. Several stock ranges are expanded or combined. Three management units are proposed for S. attenuata: the coastal, northern offshore, and southern offshore spoiled dolphins. Four management units are proposed for S. longirostris: the Costa Rican, eastern, northern whitebelly, and southern whitebelly spinner dolphins. Two provisional management units are proposed for S. coeruleoalba: the northern and southern striped dolphins. Five management units (two of which are provisional) are proposed for D. delphis: the Baja neritic, northern, central, southern, and Guerrero common dolphins. Division into management units was based on morphological stock differences and distributional breaks. (PDF file contains 34 pages.)

An analysis of migratory salmonid catch effort data, derived from anglers' log books, 1991

1000 log books were issued to anglers of which 236 were returned, those from the rivers Derwent, Kent, Lune and Ribble accounted for the vast majority. The Derwent had the highest catch rate of these rivers: one salmon every 13.89 hours followed by the Lune, Kent and Ribble at 16.39, 18.87 and 35.71 hours, respectively. For sea trout the Lune, Derwent and Ribble had a catch rate of approximately one fish every 10.0 hours (9.8, 10.0 and 10.64 hours),and for the Kent one fish per 16.1 hours fished. Salmon angling visits were, in general,longer than those for sea trout being between 2 and 6 hours as opposed to 2 to 4 hours. On the majority of visits (>80%) no fish were caught and was the same for salmon and sea trout. For salmon the majority of fish were caught on fly, spinner or worm, and the least on prawn. For sea trout fly predominated. The majority of salmon caught were less than 91b in weight and were presumed to be grilse (1 sea winter). The majority of the sea trout caught weighed between 1 and 31b. The pattern of catch, effort, CPUE, abundance and catchability for salmon and sea trout were modelled using the data from the rivers Derwent, Kent and Lune. Flow significantly influenced catch, effort and catchability of salmon which had entered in a particular month. For sea trout flow was not significantly correlated with any of the dependent variables. The catchability coefficient for salmon, determined from the total number of fish, remained relatively constant over the period June to October indicating that CPUE was a reasonable measure of within season abundance. This was not found to be the case for sea trout.

Prevalence of the commensal barnacle Xenobalanus globicipitis on cetacean species in the eastern tropical Pacific Ocean, and a review of global occurrence

Distribution and prevalence of the phoretic barnacle Xenobalanus on cetacean species are reported for 22 cetaceans in the eastern tropical Pacific Ocean (21 million km2). Four cetacean species are newly reported hosts for Xenobalanus: Bryde’s whale (Balaenoptera edeni), long-beaked common dolphin (Delphinus capensis), humpback whale (Megaptera novaeangliae), and spinner dolphin (Stenella longirostris). Sightings of Xenobalanus in pelagic waters are reported for the first time, and concentrations were located within three productive zones: near the Baja California peninsula, the Costa Rica Dome and waters extending west along the 10°N Thermocline Ridge, and near Peru and the Galapagos Archipelago. Greatest prevalence was observed on blue whales (Balaenoptera musculus) indicating that slow swim speeds are not necessary for effective barnacle settlement. Overall, prevalence and prevalence per sighting were generally lower than previously reported. The number of barnacles present on an individual whale was greatest for killer whales, indicating that Xenobalanus larvae may be patchily distributed. The broad geographic distribution and large number of cetacean hosts, indicate an extremely cosmopolitan distribution. A better understanding of the biology of Xenobalanus is needed before this species can be used as a biological tag.

Abundance of cetaceans in the oceanic northern Gulf of Mexico from 2003 and 2004 ship surveys

The Gulf of Mexico (GMx) is a subtropical marginal sea of the western North Atlantic Ocean with a diverse cetacean community. Ship-based, line-transect abundance surveys were conducted in oceanic waters (>200 m deep) of the northern GMx within U.S. waters (380,432 square km) during summer 2003 and spring 2004. Data from these surveys were pooled and minimum abundance estimates were based on 10,933 km of effort and 433 sightings of at least 17 species.The most commonly sighted species (number of groups) were pantropical spotted dolphin, Stenella attenuata (115); sperm whale, Physeter macrocephalus (85); dwarf/pygmy sperm whale, Kogia sima/breviceps (27); Risso’s dolphin, Grampus griseus (26); and bottlenose dolphin, Tursiops truncatus (26). The most abundant species (number of individuals; coefficient of variation) were S. attenuata (34,067; 0.18); Clymene dolphin, S. clymene (6,575; 0.36); T. truncatus (3,708; 0.42); and striped dolphin, S. coeruleoalba (3,325; 0.48). The only large whales sighted were P. macrocephalus (1,665; 0.20) and Bryde’s whale, Balaenoptera edeni (15; 1.98). Abundances for other species or genera ranged from 57 to 2,283 animals. Cetaceanswere sighted throughout the oceanic northern GMx, and whereas many species were widely distributed, some had more regional distributions. Compared to abundance estimates for this area based on 1996-2001 surveys, the estimate for S. attenuata was significantly smaller (P <0.05) and that for the spinner dolphin, S. longirostris, appeared much smaller. Also, P. macrocephalus estimates were based on less negatively biased estimates of group-size using 90-minute counts during 2003 and 2004.

Prawn culture and policy options: technology import and culture through fishermen vis a vis industry

Recent developments in aquaculture has created an awareness that prawn culture is a dollar spinner, in which industry can step in to earn foreign exchange by producing an expensive food iten which has a high market demand abroad. The Government has to take a policy decision whether the prawn culture should be done through small fishermen to improve their socio-economic condition or through private industry with the high technology input and predefined objectives of export trade. Perhaps a simultaneous operation of the two could be allowed best in the interest of India. Perhaps in the interest of quick development and adoption of high production technology, through fishermen organization, the development is encouraged through the implimentation of welfare and area development schemes. In some selected areas private industry may be encoureged to use high production technology to develop prawns.

Culture of green algae Chlorella ellipsoidea in inexpensive media

The culture of the of green alga Chlorella ellipsoidea was conducted under natural conditions at the same place simultaneously in five different media, viz., medium-I (inorganic medium), medium-II (powdered whole-pulse medium), medium-III (medium of pulse bran), medium-IV (mixed medium = 50% inorganic medium + 50% whole-pulse powder medium), medium-V (mixed medium = 50% inorganic medium + 50% pulse bran medium). The culture was done in 500 ml conical flask. Growth rates of C. ellipsoidea in five different media were different and reached maximum cell densities of 0.63 x 10^6 cells per ml in 8 days in medium-I, 4.02 x 10^6 cells per ml in 10 days in medium-II, 3.62 x 10^6 cells per ml in 9 days in medium-III, 4.38 x 10^6 cells per ml in 11 days in medium-IV and 4.36 x 10^6 cells per ml in 11 days in medium-V. The range of air temperature was 20 to 33°C and that of culture media was 24 to 32°C and light intensity was 2000 to 7000 lux during the culture period. The inexpensive culture media were found to be significantly useful for algal culture.

软件属性保障中的源代码静态分析技术研究

随着硬件性能的不断提升，计算机正在被赋予越来越艰巨的任务，运行其上的软件作为沟通人类思维和底层硬件的桥梁，其重要性日益增加。与此同时，软件系统的规模也在不断变大，所涉及的逻辑也更为复杂，这导致开发人员难免会由于疏漏在软件设计实现的过程中引入缺陷、埋下隐患。所以,如何检验、确保软件的属性就成为时下一个亟待解决的热点问题。而在此背景下，源代码静态分析技术由于恰好可以弥补现有测试方法的不足，已经开始在这一研究领域崭露头角。有鉴于此，本文为了推进安全信息系统的研发，分别围绕源代码静态分析技术在软件属性保障中两个最主要的应用场景展开研究，涉及高等级安全操作系统开发过程中的源代码自动化审计，以及分布式信息系统中平台间互信建立时针对软件属性所进行的远程验证，其中，前者是为从深度上将现有安全操作系统向更高等级推进提供助力，而后者是为了从广度上将信息安全领域现有的围绕单机平台的研究成果向分布式架构推广建立基础。具体来说，本文选择针对编程接口规范的一致性检验和应用静态分析的软件属性远程验证作为研究的切入点，探讨了应用源代码静态分析技术检验、确保软件属性的方法和用途，主要取得以下几个方面的成果： 第一，本文给出了一个基于值等价类的别名分析方法。该方法依据相关的传值操作维护一个值等价类空间，可以在编程接口规范一致性检验的过程中按需推导变量符号间的等值关系，不仅有能力支持上下文相关、路径相关的全局分析，还可以有效应对C代码中因结构、指针等构件所衍生出来的大量变量符号。 第二，针对大部分现有代码静态分析工具分析规模受限的问题，本文围绕编程接口规范的一致性检验给出了可以与别名分析有效结合的性能优化方案。该方案不仅能通过剔除与分析无关的执行分支和引入缓存机制提高分析效率，还可以尽量确保分析的准确性少受影响。 第三，我们设计、实现了一个C代码静态分析工具ABAZER（A Bug AnalyZER）。该工具可以依据用户使用有限自动机模型描述的编程接口规范，对操作系统内核级别的软件进行全局分析，指出代码中可能有悖于规范的部分。我们使用ABAZER实际考查了FreeBSD内核中锁机制以及GCC 4.x中库GNU Libiberty的使用情况，从中发现了若干真实的缺陷。 第四，本文针对现有应用可信计算技术、基于完整性信息进行远程验证的方案在灵活性和实用性上所存在的不足，给出一个扩展方案。该方案通过引入虚拟机技术，在软件构建过程中收集举证信息，应用静态分析方法分析软件功能模块间的相关性，划分出与验证相关的模块，有效控制用户定制软件验证时所要依赖的可信列表的规模，使其有能力适应当今网络环境中的大量异质平台和各种安全需求。此外，它还可以为自身所依赖的可信计算基的替换和更新提供支持。 第五，本文针对Flask架构的特点，给出了一个既能检验强制访问控制实现正确性，又能最大限度保留软件灵活性、使得用户可以在一定程度上对软件进行定制的远程验证方案。该方案依赖源代码静态分析技术界定软件中无需基于完整性进行验证的模块，在进一步缩减可信列表规模的同时，使用代码改写技术在这些模块中自动化地插入监控代码约束软件的动态行为，以达到确保强制访问控制实现正确性的目的。该方案初步展现了源代码静态分析技术在远程验证中广阔的应用前景。

己酸乙酯酯化真菌筛选及发酵条件研究

本文主要研究了具有己酸乙酯酯化活性的真菌菌株的筛选和发酵条件优化。从大曲和糟醅样品中分离纯化获得79株产生透明圈的丝状真菌，菌落形态初步识别结果显示分离菌株包括红曲霉属、根霉属及曲霉属等菌株。其中菌株EM-56酯化酶活力最强，发酵获得的粗酶制剂酶活为172.36 u。根据显微形态、菌落形态及生理生化特征，初步鉴定该菌株为曲霉科红曲霉属紫色红曲霉（Monascus purpureus）。 在此基础上重点研究了菌株EM-56在不同培养基成分及不同培养条件下的产酶情况，确定了最佳培养基和培养条件。通过单因素实验确定在基础培养基中添加最佳碳源为葡萄糖，最佳氮源为蛋白胨。正交优化实验结果确定了最佳培养基组成：以麸皮为基础培养基，添加葡萄糖 2%，蛋白胨 0.3 %，KH2PO4.3H2O 0.05 %，MgSO4.7H2O 0.06 %。菌株EM-56在上述培养基中的最佳发酵条件为：初始pH 5.5，发酵温度为35°C，发酵时间7d，种龄48h，接种量8%，装瓶量50g / 瓶（500mL）。在最佳培养基和发酵条件下，菌株EM-56发酵获得的粗酶制剂酶活达到241.56 u，比优化前提高了40.15%。 In this paper, the research focuses on the selection of fungus with esterifying activity and optimization of fermentation conditions. We isolated 79 strains which had transparent zones from Daqu and fermented grains. The isolated strains contained Monascus、Rhizopus and Aspergillus through primary morphology analysis. The strain of EM-56 which produces strongest esterase was selected. The enzyme activity reached 172.36u. According to related literature, EM-56 was identified as Monascus purpureus through morphology analysis and biochemical determination. We also studied the effects of different medium and fermentation conditions on the esterase production of strain EM-56. The optimal medium and fermentation conditions were determined. Single factor experiment result shows that the optimal carbon source added is glucose and the optimal nitrogen source added is peptone. The optimal fermentation medium determined by orthogonal optimization test is as follows: wheat bran as substrate, glucose 2%, peptone 0.3%, KH2PO4.3H2O 0.05%，MgSO4.7H2O 0.06%. The optimal fermentation conditions are: initial pH 5.5, cultural temperature 35°C, cultural time 7d, seed age 48h, inoculation 8%, medium mass 50g / flask（500mL）. The esterse activity of EM-56 cultivated in the optimal medium and fermentation conditions reached 241.56u and increased by 40.15% compared with the original activity.

鲜甘薯快速乙醇发酵条件的初步研究

本文对不同菌种（酵母菌和运动发酵单胞菌）快速生产燃料乙醇的条件进行了研究，实现了鲜甘薯快速转化为燃料乙醇。全文分为两部分： 第一部分：酵母菌快速生产燃料乙醇的条件研究。通过单因素试验，酵母菌快速生产燃料乙醇的条件为：发酵方式采用边糖化边发酵（SSF），蒸煮温度为85 ℃，料水比2:1（初始糖浓度 210 g/kg），糖化酶用量0.75 AGU/g 鲜甘薯，接种量10%（v/w）。在最优条件下，经过24 h发酵，乙醇浓度可达97.44 g/kg, 发酵效率为92%，发酵强度为4.06 g/kg/h。由于采用了低温蒸煮和SSF，可以大大节约能耗，从而降低乙醇生产的成本。同时，利用摇瓶优化的条件，进行了10 L，100 L，500 L发酵罐的放大试验，由于发酵罐初期可以人为通氧，使菌体能迅速积累，发酵时间缩短2 h，发酵效率在90%以上。 第二部分：运动发酵单胞菌快速生产燃料乙醇条件研究。通过单因素试验和正交试验获得了发酵的最佳参数：初始pH值6.0-7.0，硫酸铵5.0 g/kg，糖化酶量1.6 AUG/kg淀粉，初始糖浓度200 g/kg，接种量12.5%（v/w）。经过21 h发酵，乙醇浓度为95.15 g/kg，发酵效率可达94%。同时对不灭菌发酵也进行了研究，发酵效率可达92%。为鲜甘薯运动发酵单胞菌燃料乙醇的工业化生产打下基础。 对发酵结束后的残糖进行了研究。通过薄层层析和葡萄氧化酶测定证明：无论是酵母菌还是运动发酵单胞菌发酵结束后的发酵液中都不含葡萄糖。经过HPLC进一步分析残糖说明：发酵液中已没有葡萄糖成分；经糖化酶水解后仍没有葡萄糖出现；但经酸水解后又出现了葡萄糖，说明结束后的残糖是一些低聚糖结构。有关残糖的结构需要进一步研究。可以通过开发高效的低聚糖水解酶来降低发酵液的残糖，提高原料的利用率。 A new technology for rapid production fuel ethanol from fresh sweet potato by different microorganisms (Saccharomyces cerevisiae and Zymomonas mobilis) was gained in this research. The paper involved two parts: Part 1: The study on fuel ethanol rapid production from fresh sweet potato by Saccharomyces cerevisiae. The following parameters of Saccharomyces cerevisiae was investigated by a series of experiments: fermentation models, cooking temperature, initial sugar concentration and glucoamylase dosage. The results showed that SSF (simultaneous saccharification and fermentation) not only reduced the fermentation time (from 30 to 24h) but also enhanced the ethanol concentration (from 73.56 to 95.96 g/kg). With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg which the fermentation yield could reach to 92% and ethanol productivity 4.06 g/kg/h from sweet potato enzymatic hydrolysis. Furthermore, the savings in energy by carrying out the cooking (85 ℃) and saccharification (30 ℃) step at low temperature had been realized. The results were also verified in 10 L, 100 L and 500 L fermentor. The fermentation yield was no less than 90%. The fermentation time of fermenter was shorter than Erlenmeyer flask. This may be that the aeration in the early fermentation period is available, which lead to the rapidly commutations of biomass. Part 2: The technology of ethanol rapid production with simultaneous saccharification and fermentation ( SSF ) by Zymomonas mobilis，using fresh sweet potato as raw material was studied. The effects of various factors on the yield of ethanol were investigated by the single factor and the orthogonal experiments. As a result, the optimal technical conditions were obtained from those experiments：initial pH value 6.0-7.0, nitride 5.0 g/kg，(NH4)2SO4, glucoamylase 1.6 AUG/kg starch, inoculums concentration 12.5% (v/w). The Zymomonas mobilis was able to produce ethanol 95.15 g/kg, with 94% of the theoretical yield, from fresh sweet potato after 24 h fermentation. The fermentation efficiency of non-sterilized was also reach to 92%. We also analyzed the final fermentation residual sugars of Saccharomyces cerevisiae and Zymomonas mobilis. When the residual sugars were analyzed by thin-layer chromatogram and glucose oxidase, there was no glucose. The analysis of reducing sugars by HPLC showed that there was no glucose existed in the fermentation liquor. However, the glucose appeared after being hydrolyzed by acid. It is indicated that the residual sugars in the final fermentation liquor were the configuration of oligosaccharide, which was linked by the special glycosidic bonds. It was feasible for reducing residual sugars to develope the enzyme that can degradation the oligosaccharide.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

微生物法生成辅酶Q10的研究

本文根据我们实验室建立的发酵产物中辅酶Q10定性定量检测方法，筛选得到一株可以代谢产生较多辅酶Q10的野生菌株放射形土壤杆菌(Agrobacterium radiobacter No.50)。 为了提高放射形土壤杆菌的辅酶Q10的产量，本实验利用液体培养研究了单因素对菌株辅酶Q10产量的影响，并用正交法确定了最佳液态发酵条件。最佳发酵培养基是：葡萄糖20g,蔗糖40g, 硫酸铵10g,玉米浆30g, 酵母膏3g，K2HPO4 3g,MgSO4.7H2O 1g，蒸馏水1000mL,pH 7.0-7.2。最佳发酵条件是：转接斜面菌种到种子培养基, 转速220r/min、温度28。C培养24h后，转入发酵培养基(250mL三角拼装液量为50mL，pH 7.0), 接种量为10％，转速220r/min、温度28。C，培养120h。在此条件下，菌体湿重约为50g/L，辅酶Q10含量约为20mg/L。 本文以放射形土壤杆菌为出发菌株进行诱变育种，以期获得辅酶Q10高产菌。根据微生物育种原理、参照辅酶Q10的代谢调控机制，以野生型放射形土壤杆菌(Agrobacterium radiobacter No.50)为出发菌株，采用紫外线和亚硝基胍复合诱变技术，依次筛选得到菌体提取物M抗性菌ARM－7、烟草提取物T抗性菌株ARMT-26、Vk3抗性菌株ARMTV-25、链霉素抗性菌株ARMTVS-32，菌株ARMTVS-32产量达到了36.8mg/L，与原始出发菌株相比，产量提高了77％。 研究了茄尼醇、对羟基苯甲酸、橘子皮提取物D、胡萝卜提取物E、烟草提取物对ARMTVS-32合成辅酶Q10的影响，结果表明这些物质对菌体合成辅酶Q10有一定促进作用，添加0.2g/L茄尼醇时，辅酶Q10含量提高了17%，达到了40.7mg/L；添加1.2g/L橘子皮提取物D时，辅酶Q10含量提高了13.8%，达到了39.6mg/L；添加0.5g/L胡萝卜提取物E时，辅酶Q10含量提高了25.3% ，达到了43.6mg/L；添加8g/L烟草提取物时，辅酶Q10含量提高了12.6%，达到了39.2mg/L。 Production of Coenzyme- Q10 (CoQ10) by fermentation is considered as a process with broad prospects.Quantitative Analysis of CoQ10 in the culture of microbe by TLC—UV spectrophotometry was developed, by using this method we got the strain Agrobacterium radiobacter,which was isolated from forest soil of southwest of China. The effect of the single factor on CoQ10-production ability of the strain was examined by liquid cultured, and its best optimum cultivation conditions were established by orthogonal method. The results showed that the optimum fermentation conditions were as following: carbon sources glucose 20g/L,sucrose 40g/L; nitrongen sources (NH4)2SO4 10g/L,maize liquid 30g/L;yeast extract 3g; K2HPO4 3g/L,MgSO4.7H2O 1g/L; initial pH was 7 and volume of medium(medium volume vs flask volume) was 50mL/500mL, incubating for 120h on a rotary shaker at 220 rpm and 28℃.Under these conditions, the biomass and CoQ10 concentration reached 50g/L and 20mg/L respectively. According to the biosynthesis mechanism of CoQ10 and breeding theory, CoQ10 over-production strains were screened by UV--NTG. mutation using Agrobacterium radiobacter No.50 as parent strain. A microbe-juice resistant mutant ARMTVS-32, which also could resist tobacco-juice, VK3 and streptomycin, was screened out from an agar plate. The CoQ10 content of ARMTVS-32 reached 36.8mg/L, which was 77% higher than the initial strain. In addition, We discussed the effects of some organic substrates on the synthesis of CoQ10 in ARMTVS-32. The results showed that solanesol, orange juice D, carrot juice E and tobacco juice could promote the CoQ10 accumulation in the cells. The CoQ10 content of ARMTVS-32 reached 40.7mg/L when added 0.2g/L solanesol,it reached 39.6mg/L when added 1.2g/L orange juice D，it reached 43.6mg/L when added 0.5g/L carrot juice E. it reached 39.2mg/L when added 8g/L tobacco juice.

一株掷孢酵母利用红薯淀粉酶解液积累油脂研究

本论文以红薯淀粉的双酶法水解液为碳源，从19 株红色酵母中筛选出一株油脂含量较高的菌株掷孢酵母（Sporobolomyces reseus）As.2.618。为了提高掷孢酵母（S.reseus）As.2.618 的油脂产量，考察了培养基组成对该菌生长情况及油脂积累的影响。用均匀设计法对培养基组成进行了优化，由DPS软件得出的优化结果为：还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。生物量可达19.23 g/L,油脂含量为3.875 g/L。研究了添加二价离子对该菌的生长及油脂积累的影响,结果表明Zn2+对该菌生长和油脂积累都有显著促进作用。研究了发酵条件以及添加氧载体正十二烷对该菌发酵的影响，表明添加正十二烷有利用于该菌生长与油脂积累。得出最佳发酵条件是：在还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。添加30mg/L 硫酸锌,接种量为5％，在24h 后添加2g/L 的碳酸钙和2%(v/v)正十二烷，pH6.0 培养温度为27℃，转速为200r/min，培养时间为7 天的条件下，该菌生物量干重可达35.05g/L,油脂含量也达11.98g/L。Lipid is one of the basic material for life-sustaining activities andimportant industrial materials. As lipid resources mainly come from the animal andthe plant, the problem of lipid lack is encountered at times. The lipid frommicroorganisms is the substitute and superior to the above lipid with a short period ofproduction and much cheaper fermentation materials such as agricultural and sidelineproducts or wastes of crop.Thus large scale production and broad application ofmicrobial lipid will be efficient not only in substitute of the animal and the plant lipidfor food and industrial field , but also inducing a new way leading to solve the energyproblem.For the purpose of exploring the characteristics of lipid production of redyeasts from sweet potato starch hydrolysates. 19 red yeasts are screened for thecapability of lipid producing and one strain Sporobolomyces reseus As.2.618 withsuperior performance is sellected.To improve the Sporobolomyces reseus As.2.618’s capability of lipidaccumulation , the components of the medium, which may influence the growth of thestrain and the lipid yield have been studied. To get the optimum mediumcomponents ,the “uniform design” was used .The DPS software gave the optimummedium component is: reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO4 0.3g/L、MgSO4 0.15 g/L. The biomass could reach up to 19.23 g/L and lipid yield 3.87g/L with the above composition of fermentation medium.Furthermore the fermentation conditions , addition of the divalent metal ionsand the oxygen vector to increase the strain’s lipid producing capability are tested.The optimum condition is : reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO40.3 g/L、MgSO4 0.15 g/L，Adding 30mg/L ZnSO4,and adding 2g/L CaCO3 2％(v/v)n-dodecane after 24h’s fermentation. the optimal fermentation condition were asfollow :30ml medium in the 500ml flask with initial pH 6.0,the flasks with 5%inoculation volume were at 200r/min shaking speed for 7d’s fermentation at27 .Under this kind of condition the high biom ¡æ ass which reach to 35.05 g/L could begot ,the yield of lipid also could reach to 11.98g/L.