梨渣固态发酵培养多粘类芽孢杆菌的工艺

为了充分利用梨渣这一资源,该文以新鲜鸭梨渣为主要原料,采用固态发酵方式,研究了梨渣与麸皮比例、外加氮源、无机盐、装瓶量、培养温度和时间对生防用多粘类芽孢杆菌D1生长及产芽孢的影响。研究发现,梨渣是一个良好的固态发酵原料,适合培养多粘类芽孢杆菌。当梨渣与麸皮的质量比为5∶2时,获得最大活细胞数,为2.5×109 cfu/g。向梨渣发酵培养基中添加尿素、硝酸钠和豆饼粉可明显提高活菌数和芽孢数,其中添加豆饼粉的效果较好。培养基中添加质量分数为0.5%磷酸二氢钾极大地促进了菌体生长和芽孢的形成。随着培养基装瓶量的增加,芽孢数也随之明显减少。多粘类芽孢杆菌D1的较适培养温度为34°C,较适培养时间为120 h。适宜培养条件下的活菌数、芽孢数和芽孢率分别为5.09×109 cfu/g、4.78×109 cfu/g和93.9%。该研究结果为利用梨渣发酵生产多粘类芽孢杆菌菌剂提供参考。     

多粘类芽孢杆菌BRF-1和枯草芽孢杆菌BRF-2对黄瓜和番茄枯萎病的防治效果

采用室内培养方法,对生防细菌多粘类芽孢杆菌BRF-1和枯草芽孢杆菌BRF-2不同稀释倍数的代谢产物抑制黄瓜尖孢镰刀菌、番茄枯萎病菌分生孢子萌发和菌丝生长的试验结果表明,当两菌株代谢产物稀释5倍时,对分生孢子萌发的抑制率达到80％以上;两菌株代谢产物在稀释2倍和5倍时,对2种病原真菌菌丝生长的抑制率在40％-80％之间,与对照差异极显著（P〈0．01）。盆栽试验表明,BRF-1和BRF-2生防细菌菌悬液及其无菌代谢物对黄瓜和番茄枯萎病不仅具有较好的防治效果,而且具有明显的促进生长作用。     

侧胞芽孢杆菌发酵工艺的研究

侧胞芽孢杆菌是应用于微生物肥料的一种重要功能菌,采用正交试验筛选了侧胞芽孢杆菌的发酵培养基配方,并探讨了发酵工艺条件。结果表明,侧胞芽孢杆菌的摇瓶发酵最佳培养基为蔗糖3.0%、酵母膏0.8%、蛋白胨1.2%、MgSO40.075%、KH2PO40.25%、MnSO40.010%、CaCO30.8%最佳;培养条件为温度32.5℃、转速200 r/min、接种量2%以上、pH为7.6,发酵液中的所得菌浓度可达9×108个/mL。     

外包木霉内含芽孢杆菌颗粒生物有机肥研制及其促生效应

  目的  利用造粒喷涂技术,研制了外包木霉菌内含芽孢杆菌（SQR9）的颗粒复合菌生物有机肥,以辣椒为供试作物,利用盆栽试验,研究了新型生物有机肥对辣椒植株株高、茎粗、叶绿素等农艺性状的影响。  方法  首先利用圆盘造粒机将有机肥、芽孢杆菌和适量粘合剂制成颗粒芽孢杆菌生物有机肥,随后,再将真菌木霉孢子喷涂颗粒表面,利用第一次添加的粘结剂形成外包真菌孢子膜的新型颗粒复合菌生物有机肥。  结果  货架期试验结果表明,颗粒肥料中木霉菌比粉状肥料存活能力更强,在储存60天后,新型颗粒复合菌生物有机肥中木霉菌含量大于2 × 107 cfu g?1,芽孢杆菌数量大于1.2 × 108 cfu g?1。盆栽试验结果表明,在种植47天后,施用新型颗粒复合菌生物有机肥的辣椒植株在株高、茎粗、叶绿素、鲜重和干重方面均高于其他处理（含SQR9颗粒生物有机肥、含SQR9和木霉菌的粉状生物有机肥、含木霉菌颗粒生物有机肥和含SQR9和木霉菌的颗粒生物有机肥处理）,表明了该新型颗粒复合菌生物有机肥对辣椒具有明显的促生效果。  结论  相比于单菌和其他工艺研制的复合菌生物有机肥,外包木霉真菌内含芽孢杆菌新型颗粒复合菌生物有机肥能够充分发挥木霉真菌和芽孢杆菌的促生功能,有效增强复合菌生物有机肥的促生效果。     

多粘类芽孢杆菌JSa-9高产LI-F类抗菌脂肽突变株的选育

为了提高多粘类芽孢杆菌JSa-9产LI-F类抗菌脂肽的产量,本研究采用亚硝基胍(NTG)、60Co-γ射线和低能N+离子注入3种不同诱变方法,确定了NTG最佳诱变浓度为50μg·mL-1,60Co-γ射线最佳诱变剂量为200Gy;当低能N+离子注入能量为10keV,最佳注入时间为60 s,当注入能量为20keV,最佳注入时间为30 s。最终从300株突变株中筛选得到1株苯丙氨酸缺陷型菌株N1-37和1株组氨酸缺陷型菌株N2-27,均由NTG诱变获得。以金黄色葡萄球菌为指示菌进行抑菌试验,N1-37和N2-27抑菌圈直径比出发菌株JSa-9分别增加45.54%和32.35%。通过高效液相色谱确定LI-F类抗菌脂肽产量,结果表明,N1-37和N2-27产LI-F类抗菌脂肽的产量是原始菌株JSa-9的1.71倍和1.4倍,经连续传代10代,试验表明2菌株遗传稳定性良好,不仅可以应用于工业生产上,还可以作为亲本菌株进行细胞融合研究。     

多粘类芽孢杆菌LRS-1对辣椒疫霉病害根际土壤细菌多样性的影响

【目的】研究多粘类芽孢杆菌LRS-1生防菌对患疫霉病的辣椒根际土壤细菌多样性的影响。【方法】通过对无病阴性对照(CK)、辣椒疫霉菌阳性处理(PC)和生防菌+辣椒疫霉菌处理(LRS1) 3个处理的盆栽根际土壤样品16S r RNA基因的V3-V4区进行高通量测序,并对下机数据予以相关生物信息学分析,获得了不同处理根际土壤细菌的OTU丰度、Alpha多样性、OTUs分布、群落组成差异以及PCA聚类分析等数据结果。【结果】各处理间的根际土壤细菌群落变化明显,疫霉菌处理明显降低了根际土壤细菌群落的丰富度和多样性,而接种多粘类芽孢杆菌LRS-1则可明显改善根际土壤细菌群落丰富度与多样性;接种LRS-1明显提高了辣椒根际土壤细菌种类组成的相似性,并改善疫霉菌胁迫下的细菌种类组成;各处理的优势物种类群均分布于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria),但疫霉胁迫条件下Sphingomonadaceae、Clostridiales物种丰度显著降低,而Burkholderiales、Micrococcales等物种丰度显著增加,接...     

巨大芽孢杆菌P1的解磷效果与发酵条件研究

以巨大芽孢杆菌P1为研究对象,利用钼锑抗比色法测定了菌株的解磷能力,采用单因子实验及正交实验优化了菌株的最佳发酵条件,并在10 L发酵罐上进行了发酵条件验证。巨大芽孢杆菌P1在蛋黄培养基上可产生明显的溶磷圈,溶磷圈直径和菌落直径的比值D/d=4.5。在卵磷脂液体培养基中培养4 d后,上清液中的有效磷含量为55.66 mg/mL,是对照的101.2倍,说明溶磷效果明显。单因子实验表明,最佳培养温度为32℃,培养时间为32 h,培养基初始pH为7.5。正交实验表明,最佳培养基组成为玉米粉20 g,黄豆粉10 g,K2HPO41.5 g,MgSO4.7H2O 1.5 g,CaCO31.5 g,H2O 1 000 mL,pH 7.5。方差分析表明,玉米粉、黄豆粉在α=0.05水平上对实验结果的影响存在显著性差异。10 L发酵罐发酵结果表明,巨大芽孢杆菌P1发酵32 h基本达到终点,菌数达到60.0×108cfu/mL,芽孢比率达90%以上。     

枯草芽孢杆菌发酵制备花生粕饲料条件优化

长期以来花生粕作为动物饲料得到广泛的应用,为提高其利用价值本文以热榨花生粕作为固体发酵培养基,以加水量、接种量、发酵时间、发酵温度、麸皮含量为变量进行单因素试验,采用响应面分析试验对枯草芽孢杆菌发酵制备花生粕饲料的条件进行优化。结果表明:枯草芽孢杆菌发酵制备花生粕饲料的最佳条件是花生粕18g、加水量20mL、接种量0.44g、发酵时间60h、发酵温度30℃。在此条件下,发酵后产物芽孢杆菌芽孢数较高。     

接种贝莱斯芽孢杆菌SW5菌株对发酵鳀鱼鱼露品质的影响

为探讨接种贝莱斯芽孢杆菌SW5菌株对发酵鳀鱼鱼露的影响,以贝莱斯芽孢杆菌SW5为唯一发酵菌株,以低值鳀鱼为原料,测定鱼露发酵过程中氨基酸态氮(AA-N)、挥发性盐基氮(TVB-N)、pH值、总酸含量,并使用顶空固相微萃取结合气相色谱-质谱联用仪(HS-SPME-GC-MS)测定发酵结束后发酵液的挥发性风味物质。结果表明,在发酵期间,利用SW5菌株发酵的处理组1在发酵第6天时AA-N含量最高,为0.76 g·100 mL^-1,达到市售二级鱼露标准。3种处理组和舟山商品鱼露中共检测出挥发性风味成分82种,主要是醇、酸、醛、酮、呋喃、烷烃及其他类化合物,接种组中的挥发性风味物质(40种)较未接种组(35种)有所增加。综上,接种贝莱斯芽孢杆菌SW5菌株发酵鱼露,能缩短发酵时间,增加风味物质种类,该菌株可用作海洋蛋白质源发酵精深加工的优良微生物菌株。     

多粘类芽胞杆菌菌株M-1启动子片段的克隆及绿色荧光蛋白的表达

应用启动子探针载体pECE7,从多粘类芽胞杆菌（Panebacillus polymyxa）菌株M-1基因组克隆到一系列启动子活性片段,通过氯霉素（chloramphenicol chloromycetin,Cm）浓度梯度筛选出Cm抗性较强的3个片段。将这3个具有启动子活性的片段与来自GFP表达载体pGFP78的gfpmut3a基因插入Escherichiacoli-Bacillus穿梭载体pHY300PLY中,获得GFP表达载体pGFP5,pGFP8和pGFP17。通过热激转化E.coliDH5α,荧光显微镜下观察到明亮的绿色荧光,表明筛选到的启动子片段具有良好的启动外源基因gfp转录的功能。同时利用此表达载体标记本实验筛选得到的生防蜡样芽胞杆菌（Bacilluscereus）菌株B905,获得了GFP标记的工程菌Bacillus cereus B905（gfp-5、gfp-8和gfp-17）,通过荧光显微镜观察,发现转入GFP表达载体pGFP5和pGFP8后,菌体有明亮的绿色荧光,而转入pGFP17的菌体荧光相对较弱。     

Ex situ genetic conservation of endangered Vatica guangxiensis (Dipterocarpaceae) in China

RAPD polymorphisms were applied to check the efficiency of ex situ genetic conservation of endangered Vatica guangxiensis X. L. Mo. endemic to southwestern China. Low level of genetic variation was revealed in three remaining natural populations. Twenty random primers, each with 10 base pairs, generated 231 bands with 53.68% being polymorphic, and with an average of 32.46% being polymorphic in each natural population. Strong population differentiation was revealed by AMOVA (analysis of molecular variance) and Gst value was 0.3764. The population ML ex situ conserved in the Xishuangbanna Tropical Botanical Garden contained an intermediate genetic variation compared with natural populations, with 30.74% bands being polymorphic. Of the total 231 bands generated in V. guangxiensis, 204 bands were also detected in population ML, indicating that 88.31% of the total genetic variations of this species were conserved in ex situ population. If only the alleles with moderate to high frequency (P>0.05) were considered, 204 out of 209 bands (97.61%) occurred in ex situ population ML. RAPD analysis also detected one exclusive band in natural population NS, and five in natural population NP, three of these exclusive bands were generated in every samples of natural population (NP), and other three had moderate to high frequencies. While none of these exclusive bands were detected in ex situ conserved population ML. Our conclusions are that the ex situ conserved population ML contains representative genetic variation to maintain long-term survival and evolutionary process of V. guangxiensis, and that more extensive ex situ sampling in natural population NS and NP is needed to conserve more exclusive alleles in ex situ population. The tropical area in the Botanical Garden would play a more important role in the ex situ conservation of rare and endangered plants.     

Grazing of a common species of soil protozoa (Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice (Oryza sativa L.)

We performed a controlled experiment with rice seedlings (Oryza sativa L.) growing in Petri dishes on homogeneous nutrient agar containing a simple rhizosphere food web consisting of a diverse bacterial community and a common soil protozoa, Acanthamoeba castellanii, as bacterial grazer. Presence of amoebae increased bacterial activity and significantly changed the community composition and spatial distribution of bacteria in the rhizosphere. In particular, Betaproteobacteria did benefit from protozoan grazing. We hypothesize that the changes in bacterial community composition affected the root architecture of rice plants. These effects on root architecture affect a fundamental aspect of plant productivity. Root systems in presence of protozoa were characterized by high numbers of elongated (L-type) laterals, those laterals that are a prerequisite for the construction of branched root systems. This was in sharp contrast to root system development in absence of protozoa, where high numbers of lateral root primordia and short (S-type) laterals occurred which did not grow out of the rhizosphere region of the axile root. As a consequence of nutrient release from grazed bacteria and changes in root architecture, the nitrogen content of rice shoots increased by 45% in presence of protozoa. Our study illustrates that interactions over three trophic levels, i.e. between plants, bacteria and protozoa significantly modify root architecture and nutrient uptake by plants.     

Variability of communities and physiological characteristics between free-living bacteria and attached bacteria during the PAH biodegradation in a soil/water system

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) via free-living and attached micro-organisms in soil/water systems was observed in order to examine the variability in the community dynamics and physiological profiles of the micro-organisms. As determined by fluorescence in situ hybridization (FISH), the Domain Bacteria, consisting of three phyla α-, β- and γ-Proteobacteria, reached 41.27–56.05% of all organisms in the soil/water system for PAH biodegradation. Among the free-living species, Proteobacteria, including Brevundimonas (Pseudomonas) diminuta, Caulobacter spp., Mycoplana bullata, Acidovorax spp. and Pseudomonas aeruginosa were found to be dominant—making up 93.51–99.80% of the population—and therefore seem to be associated with PAH biodegradation. Total plate count numbers and the count of Pseudomonas sp. present in the free-living population increased to between 10³ and 10⁶ CFU ml⁻¹ when clay with very low organic matter content was used as the matrix for PAH degradation. However, total plate count microbial numbers increased to only 10¹–10² CFU ml⁻¹ using natural soil from Taichung containing 1.883% organic matter. The soil organic content (SOM) seemed to affect the mass transfer of PAH in soil, leading to the difference in PAH biodegradation. Two different approaches, which included community-level physiological profiling (CLPP) and ectoenzymatic activities, were used to explain the functional diversity between free-living and attached bacteria. The free-living and attached bacterial communities from the clay system showed proportionately greater differences using CLPP. Relatively high levels of esterases, aminopeptidases and some specific glycolysis-gluconeogenesis enzymes gave an identifiable correlation with PAH biodegradation. The differences in bacterial composition, numbers and physiological characteristics show that free-living and attached micro-organisms may play different biochemical roles in PAH degradation in soil.     

Suicide vectors for the introduction of genetic markers into Bradyrhizobium strains by site-directed chromosomal integration between repeated sequences (RS-α)

To achieve stable expression of the heterologous and reporter genes in the Bradyrhizobium genome, we constructed suicide plasmids capable of site-directed genomic integration of the gusA, gfp and nifA genes by homologous recombination into non-essential repeated sequences (RS-α), isolated from B. japonicum strain CPAC7 (SEMIA5080). In this report, we describe the strategies to construct the vectors and their use to obtain mutants with site-specific insertions.     

荧光原位杂交技术检测土壤中博德特氏菌探针的设计与应用

根据博德特氏菌(Bordetellasp.)的16S rRNA基因序列,设计荧光原位杂交(FISH)检测博德特氏菌的寡核苷酸探针FW_iso_62和FW_iso_761,在20%～60%甲酰胺均有很强的荧光信号。采用探针FW_iso_62及其竞争探针,结合Nycodenz和DAPI技术,建立定量检测土壤中博德特氏菌的DAPI-FISH方法。该方法可排除土壤颗粒的自动荧光对细菌信号的掩盖,保证图片中有大量微生物供统计分析,还能有效保存微生物的原位信息。应用该方法分析土壤中1,2,4-三氯苯降解菌-博德特氏菌,结果未受氯苯污染的农田土壤中没有检测到博德特氏菌,而氯苯污染土壤中检测到大量的博德特氏菌,每克土壤含3.78×106个。将该污染土壤中分离的博德特氏降解菌及其降解菌群接种至农田土壤中,降解菌的数量均随培养而增加,一个月后分别占DAPI计数的1.7%和3.8%。本研究设计的探针可有效用于复杂环境样品中博德特氏菌的定性与定量检测。     

Prey depletion caused by overfishing and the decline of marine megafauna in eastern Ionian Sea coastal waters (central Mediterranean)

Surveys primarily aimed at determining dolphin encounter rates were conducted from small inflatable craft in eastern Ionian Sea coastal waters between 1997 and 2004. During 633 surveys totalling 21,276 km of effort, observations of cetaceans and other marine species spotted in a study area of 480 km² were systematically recorded. Common dolphin encounter rates declined 25-fold across the study period, steadily decreasing from 2.18 encounters/100 km in 1997 to 0.09 encounters/100 km in 2004. Encounter rates of tuna also declined significantly. Swordfish encounter rates dropped from 1.03 encounters/100 km in 1997 to 0-0.12 in 1998-2004. Encounter rates of bottlenose dolphins did not show significant trends. The decline of high-order marine predators feeding on epipelagic prey was consistent with the hypothesis of prey depletion, likely resulting from intensive exploitation of local fish stocks, particularly anchovies and sardines. The catholic feeding habits and opportunistic behaviour of bottlenose dolphins may allow them to withstand the effects of overfishing at their present low density.     

Competition for inorganic and organic N by blue oak (Quercus douglasii) seedlings, an annual grass, and soil microorganisms in a pot study

Sources of competition for limited soil resources, such as nitrogen (N), include competitive interactions among different plant species and between plants and soil microorganisms (microbes). To study these competitive interactions, blue oak seedlings (Quercus douglasii) were grown alone or grown together with an annual grass, wild oats (Avena barbata) in pots containing field soil. We injected ¹⁵N-labeled ammonium, nitrate or glycine into the soil of each pot and harvested plants 5 days later. Plant shoots and roots, soil microbial N and soil KCl-extractable N were analyzed for ¹⁵N content. When oak and grass were grown together, ¹⁵N recovery from the inorganic N treatments (NH₄⁺, or NO₃⁻) was 34, 9 and 4% for the grass, microbes and oak seedlings, respectively, and only 1% remained as KCl-extractable N. ¹⁵N recovery from the glycine treatment was 18, 22, 5% for the grass, microbes and oak seedlings, respectively, and 4% remained as KCl-extractable N. When oaks were grown alone, ¹⁵N recovery by soil microbes was 21, 48 and 40% in the NO₃⁻, NH₄⁺ and glycine treatments, respectively. N forms had no effects on ¹⁵N recovery in oak seedlings (7%) and in KCl-extractable N pool (13%). In general, total N recovery by the grass was much greater than by oaks. However, on a fine root surface area or length basis, oaks exhibited higher N uptake than the grass. Our results suggest that the high rooting density and rapid growth rate of the annual grasses such as Avena barbata made them superior competitors for available soil N when compared to blue oak seedlings and to microbes. Soil microbes were better competitors for organic than inorganic N when annual grasses were present, but preferred NH₄⁺ when competing only with oak seedlings.     

Correlates of abundance and breeding success in the globally threatened Hinde's babbler (Turdoides hindei) and its congener, northern pied babbler (T. hypoleucus)

We investigated the relationship between habitat quality, abundance, group size and breeding success in the threatened Kenyan endemic Hinde's babbler (Turdoides hindei; Family Timaliidae), a co-operative breeder mainly associated with riverine thickets in cultivated areas. At two intensively cultivated sites, significantly more Hinde's babbler groups occurred in areas retaining at least 3% thicket cover, while breeding outcome improved where cover exceeded 9%. Despite previous concerns over low breeding success, the percentage of young birds present (17%, n=424 birds aged) was similar to that of a sympatric congener, the northern pied babbler (T. hypoleucus) (12%, n=162), and comparable with that of two other African congeners. Although breeding outcome was positively correlated with adult group size, there was no evidence that larger groups monopolised more favourable habitat or defended larger territories. Group density showed a positive, non-linear relationship with adjacent cover, levelling out where this exceeded 15-20%. Our findings suggest that even a modest increase in thicket cover may significantly raise the density and productivity of Hinde's babblers at these sites.     

Stable carbon isotope ratios of water-extractable organic carbon affected by application of rice straw and rice straw compost during a long-term rice experiment in Yamagata,Japan

ABSTRACT

Hot-water- and water-extractable organic matter were obtained from soil samples collected from a rice paddy 31 years after the start of a long-term rice experiment in Yamagata, Japan. Specifically, hot-water-extractable organic carbon and nitrogen (HWEOC and HWEON) were obtained by extraction at 80°C for 16 h, and water-extractable organic carbon and nitrogen (WEOC and WEON) were obtained by extraction at room temperature. The soil samples were collected from surface (0–15 cm) and subsurface (15–25 cm) layers of five plots that had been treated with inorganic fertilizers alone or with inorganic fertilizers plus organic matter, as follows: PK, NPK, NPK plus rice straw (RS), NPK plus rice straw compost (CM1), and NPK plus a high dose of rice straw compost (CM3). The soil/water ratio was 1:10 for both extraction temperatures. We found that the organic carbon and total nitrogen contents of the bulk soils were highly correlated with the extractable organic carbon and nitrogen contents regardless of extraction temperature, and the extractable organic carbon and nitrogen contents were higher in the plots that were treated with inorganic fertilizers plus organic matter than in the PK and NPK plots. The HWEOC and WEOC δ¹³C values ranged from ?28.2% to ?26.4% and were similar to the values for the applied rice straw and rice straw compost. There were no correlations between the HWEOC or WEOC δ¹³C values and the amounts of HWEOC or WEOC. The δ¹³C values of the bulk soils ranged from ?25.7% to ?23.2% and were lower for the RS and CM plots than for the PK and NPK plots. These results indicate that HWEOC and WEOC originated mainly from rice plants and the applied organic matter rather than from the indigenous soil organic matter. The significant positive correlations between the amounts of HWEOC and HWEON and the amount of available nitrogen (P < 0.001) imply that extractable organic matter can be used as an index for soil fertility in this long-term experiment. We concluded that the applied organic matter decomposed more rapidly than the indigenous soil organic matter and affected WEOC δ¹³C values and amounts.