高效解磷细菌的筛选及其对玉米苗期生长的促进作用

采用改良后的PVK平板,从石灰性土壤上长势良好的野生植物根表分离到44株解磷细菌,通过NBRIP液体摇瓶实验,培养7 d后发现：K₃菌株培养液中全磷浓度高达643.2 μg·ml^-1,可溶性磷为584.8 μg·ml^-1,约有12.9%的磷酸三钙被溶解出来,为对照(CK)的10.5倍;K₉菌株培养液的全磷浓度为608.5 μg·ml^-1,可溶性磷浓度为606.4 μg·ml^-1.盆栽试验的结果表明：接种解磷细菌的处理玉米株高、茎粗和干质量显著高于CK;将有机肥作为载体和解磷细菌一同混合施入土壤的处理,玉米苗干质量较单施解磷菌显著增加.经初步鉴定, K₃、K₉为假单胞菌属.     

麦田土壤解无机磷细菌的分离、筛选及其解磷效果

通过选择性解磷细菌培养基从麦田土壤中分离、纯化解无机磷细菌,并用钼蓝比色法定量研究磷细菌的解磷效果。结果表明通过选择性平板分离,发现麦田土壤中有15个菌株有解无机磷的作用,根据菌株直径和其解磷圈的大小,确定解磷能力较强的菌株9个;经过进一步分离纯化,选出单一无杂菌的菌株7个。解磷菌株经摇瓶培养后,比色测定培养液中磷的含量,通过比较发现其中5个菌株的解磷效果要显著优于其它两个菌株（P=0.05）。     

磷尾矿土壤中解磷细菌的筛选及解磷能力的测定

以贵州瓮福磷尾矿土壤为原料,从中分离纯化得到具有较高解磷能力的细菌。利用溶磷圈试验筛选出具有明显溶磷圈的细菌,通过形态学特征、生理生化试验、16S rDNA基因序列分析及系统发育树对其进行初步鉴定。再以磷酸三钙为唯一磷源对筛选所得菌株进行液态培养,探索不同菌株解磷能力与培养液pH值之间的关系,并通过钼锑抗比色法测定3株细菌的最大解磷能力。从分离纯化得到的4株细菌筛选出具有明显溶磷圈的细菌PSB1、PSB3和PSB4,初步鉴定菌株PSB1为普城沙雷氏菌(Serratia plymuthica),PSB3为嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia),PSB4为泡囊短波单胞菌(Brevundimonas vesicularis),最大解磷能力分别为148.87μg/mL、153.84μg/mL和146.76μg/mL。与国内已报道的文献相比,实验筛选所得的3株细菌都是具有较高解磷能力的菌种。菌株PSB1和PSB4培养液中pH值与其解磷能力存在显著的负相关性,而菌株PSB3不存在,其解磷机理还需进一步研究。     

棉花根际解磷菌的解磷能力和分泌有机酸的初步测定

利用特殊培养基对盐碱地棉花根际解磷菌进行了分离以及pH值和分泌有机酸能力的初步测定。利用溶磷圈法筛选出10个解磷能力较高的菌株进行深入研究,其中液体培养条件下测定了菌株的溶磷能力,有效磷在4.04~185.63 mg/L,其中wpL2溶磷量达到185.63 mg/L;测定了培养液pH值,下降到5.12~6.67,但是pH与溶磷量之间没有线性关系,测定了培养液的有机酸含量,菌株溶磷量与有机酸总量没有线性相关性,其中所分离到的解无机磷菌株均可以分泌酒石酸,除此之外,wpc1还分泌乙酸,wpc2和wpL2还分泌柠檬酸;分离到的解有机磷菌株均可分泌乙酸,除此之外,ypL1和ypc2分泌酒石酸,ypL3分泌柠檬酸,ypL2和ypc3分泌柠檬酸和丁二酸,均不能产生苹果酸。     

细菌解磷能力测定方法的研究

选择分解有机磷能力较强的3株细菌和溶解磷矿粉较强的4株细菌,砂培4周后,用不同的方法测定水浸提液中磷的含量。发现不同的细菌解磷能力差异很大,细菌在分解磷化合物的同时,一部分磷被细菌同化,一部分以无机磷酸盐状态贮藏在细菌细胞内。直接测定浸提液中无机磷酸盐的含量,将大大低估细菌的解磷能力,必须将浸提液消煮,才能比较正确地反映细菌分解磷的能力。     

青藏高原多年冻土区解磷菌筛选及抗逆能力评价

【目的】本研究旨在从青藏高原多年冻土区土壤中分离筛选高效解磷菌,为青藏高原土壤中难利用性磷的活化提供菌株资源。【方法】以青藏高原唐古拉山口附近的多年冻土区土壤为研究对象,利用有机磷和无机磷选择性培养基通过平板划线法分离解磷菌,通过16S rRNA基因系统发育分析对菌株进行鉴定,并对解磷菌的解磷能力和抗逆能力进行评价。【结果】分离筛选的5株解磷菌应归于假单胞菌属(Pseudomonas),包括3株解无机磷菌(i5、i6、i9L)和2株解有机磷菌(Qb和Qo);30 ℃培养条件下,Qb和Qo上清液有效磷含量分别为534.8 mg/L和723.7 mg/L,显著高于i5、i6、i9L上清液有效磷含量(166.9–210.5 mg/L);利用不同浓度的聚乙二醇(PEG6000)对菌株进行模拟干旱培养,5株解磷菌在PEG6000处理下均可正常生长,而且Qo上清液有效磷含量最高(519.7–683.0 mg/L);不同培养温度下,Qb和Qo在5 ℃和10 ℃培养下解磷能力要强于其他菌株的解磷能力。【结论】菌株Qo的耐低温和干旱能力强于其他4株菌,是青藏高原等高寒地区菌肥开发和植被恢复研究重要菌种资源。     

细菌解磷能力测定方法的研究*

选择分解有机磷能力较强的 3株细菌和溶解磷矿粉较强的 4株细菌 ,砂培 4周后 ,用不同的方法测定水浸提液中磷的含量。发现不同的细菌解磷能力差异很大 ,细菌在分解磷化合物的同时 ,一部分磷被细菌同化 ,一部分以无机磷酸盐状态贮藏在细菌细胞内。直接测定浸提液中无机磷酸盐的含量 ,将大大低估细菌的解磷能力 ,必须将浸提液消煮 ,才能比较正确地反映细菌分解磷的能力。     

油松菌根际高效解磷钾细菌筛选与鉴定

本研究筛选出油松-褐环乳牛肝菌菌根根际土中高效解磷钾细菌并对其进行种类鉴定。采用平板稀释法自菌根际土中筛选解磷钾细菌,并用钼蓝比色法和原子吸收法分别对其液体培养5 d后的菌株进行解磷能力和解钾能力测定,筛选出高效的解磷钾细菌。同时,对筛选出的菌株进行显微观察、生物学鉴定和16Sr DNA序列分析。通过筛选得出解有机磷细菌P6和解无机磷细菌P15能力较强,其培养液中有效磷浓度分别为283 mg/L和898.5 mg/L,经鉴定P6属于节杆菌属(Arthrobacter sp.),P15为荧光假单胞菌(Pseudomonas fluorescens),以及2株能力较强的解钾细菌K1和K12,其培养液中有效钾浓度分别为21.6 mg/L,19.3 mg/L,经鉴定K1为壤霉菌属(Agromyces cerinus),K12为中华根瘤菌属(Sinorhizobium sp.)。由此可见,油松-褐环乳牛肝菌菌根根际存在高效解磷钾细菌,褐环乳牛肝菌可以通过影响根际解磷钾细菌的种类及能力来改善油松幼苗的根际磷钾营养状况。     

三株土壤解磷细菌的分离及其解磷效果分析

旨为解决农业面源污染问题,分离鉴定土壤解磷微生物并开发复合微生物菌剂。以有机磷农药及无机难溶磷作为筛选磷源,对土壤中具有解磷能力的微生物进行分离、鉴定并对其解磷效果进行分析。从土壤中分离得到3株解磷细菌,分别命名为菌株W、Y、B;3个菌株均是革兰氏阴性菌;W菌株对敌百虫的降解能力最强,达到17.39%,B菌株对毒死蜱的降解率最强,为23.06%;3个菌株对固态难溶磷的解磷效果显著,其中B菌株解磷量最高,为96.31 mg/L;复合菌的解磷效果明显优于单菌,另外复合菌对稻田、大棚土壤解磷的促进效果显著,分别增加18.38 mg/L、14.08 mg/L。分离得到3株有效土壤解磷细菌,有一定的有机磷农药降解能力,对无机磷溶解效果较强,构建的复合菌剂对土壤解磷的促进效果显著。     

一株高效解无机磷细菌BS06的鉴定及其解磷能力分析

【目的】对一株来源于广西甘蔗根际土壤的高效解无机磷细菌BS06的分类和解磷能力进行探讨,以期为解磷微生物在广西甘蔗生产上的开发和应用提供理论依据。【方法】通过形态观察、生理生化测定及16S rRNA基因序列同源性分析,进一步结合种特异的recA基因序列分析对解磷菌BS06进行分类鉴定;通过改变无机磷培养基中的碳源、氮源对菌株解磷能力的影响,分析菌株的解磷特性;通过盆栽试验了解菌株对甘蔗品种粤糖00236、桂糖28磷素吸收的影响。【结果】分类鉴定结果表明菌株BS06属于洋葱伯克霍尔德菌(Burkholderia cepacia);菌株在以乳糖为碳源条件下具有较强的解磷能力,其发酵液中水溶性磷含量为262.71 mg/L;在以硝酸钠为氮源条件下有较强解磷能力,其发酵液中水溶性磷含量达到305.85 mg/L;接种BS06菌株显著促进甘蔗组培苗的生长并提高甘蔗植株的含磷量。【结论】解磷细菌BS06具有较大的开发利用潜力。     

溶磷真菌的筛选及耐盐特性分析

【背景】土壤盐渍化已成为影响土壤质量和作物产量的重要因素之一,利用微生物改良盐渍化土壤是既经济又环保的方法。【目的】从不同土壤样品和生物肥料中筛选溶磷能力较强的真菌并讨论其耐盐能力,为盐渍化土壤改良提供菌种资源。【方法】采用平板培养法筛选有一定溶磷能力的真菌,经ITS序列分析初步确定菌株的分类地位。以溶磷能力为考察指标,以NaCl梯度和磷酸钙梯度为考察因素,分析不同菌株利用无机磷源的能力,以及溶磷能力与pH的关系。【结果】共筛选得到16株具有较强溶磷能力的真菌,其中4株真菌对水稻发芽有显著的促进作用,它们是1株长枝木霉(MF-1)和3株踝节菌属菌株(SD-2、XJ-7和HLJ-3)。菌株SD-2和XJ-7生长的耐盐能力显著好于菌株MF-1和HLJ-3。当NaCl浓度为5%时,菌株XJ-7的溶磷能力最好,溶磷率可达9.50%;当NaCl浓度为1%时,菌株HLJ-3的溶磷能力较好,溶磷率为6.93%;当NaCl浓度为0时,菌株SD-2和MF-1的溶磷能力较强,溶磷率分别为9.07%和3.73%。进一步研究发现踝节菌属真菌的溶磷能力与菌液pH呈显著负相关关系。【结论】筛选获得的4株真菌其溶磷能力在不同盐环境中有显著差异,为今后土壤改良和生物肥料中菌种的选择提供理论依据和试验基础。     

Influence of genetic background and media components on the development of mouse embryos in vitro

One-cell embryos from some inbred and random-bred mice, but not those derived from certain F1 hybrids, suffer from a block during in vitro development known as the two-cell block. This two-cell block can be overcome by removing glucose or inorganic phosphate from the culture system or by altering the ratio of other medium components such as sodium, potassium, or bicarbonate. This issue is made more complex by the fact that the rate of development is different for each strain of mouse and this rate of development is invariably slowed under in vitro culture conditions. This study investigated the role of glucose and inorganic phosphate, individually or in combination, in relation to the two-cell block, and rate of development in vitro of two random-bred strains (CF-1 and CD-1) and an F2 hybrid derived from a nonblocking F1 hybrid cross (C57B1/6NCr × C3H/HeNCr). Results were compared with in vivo data for each strain, and between media. There was a significant difference in the rate of preimplantation development in vivo of the three strains chosen, which was mirrored in vitro, regardless of the medium. The two random-bred strains suffered from a glucose-related two-cell block which was primarily mediated by inorganic phosphate. Inorganic phosphate was detrimental to embryo development regardless of strain or the presence of glucose. Although glucose, in the absence of inorganic phosphate, resulted in some blocking in development in the inbred strains initially, its presence in media was associated with increased rates of development at later stages in embryos that did not block. Glucose, but not inorganic phosphate, was beneficial but not essential to the development of the F2 embryos. The results of this study demonstrated that mouse embryos from different strains have differential rates of development in vivo and in vitro, and different sensitivities to glucose and inorganic phosphate. The two-cell block was primarily induced in the combined presence of glucose and inorganic phosphate. Glucose was beneficial in the absence of inorganic phosphate, and inorganic phosphate was detrimental to the rate of in vitro development. © 1996 Wiley-Liss Inc.     

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making.     

Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces, and a new alanine-containing ferrichrome from Cryptococcus melibiosum

An examination of 142 strains within 19 genera of yeasts and yeastlike organisms for formation of hydroxamic acids in low-iron culture showed production of hydroxamates by two unclassified strains and by 52 strains among the genera Aessosporon (3 of 3 strains), Cryptococcus (1 of 43), Leucosporidium (3 of 11), Rhodosporidium (4 of 4), Rhodotorula (27 of 39), Sporidiobolus (2 of 2), and Sporobolomyces (12 of 13). Crystalline rhodotorulic acid was isolated in amounts sufficient to account for most or all of the measured hydroxamate in culture supernatants of 16 strains representative of the five last-mentioned hydroxamate-producing genera. A new alanine-containing ferrichrome was isolated from one strain of Cryptococcus melibiosum. Rhodotorulic acid was a major metabolic product of many of the positive strains when grown in low-iron media, and iron was shown to repress its synthesis and excretion into the culture medium. The taxonomic significance of production of hydroxamic acids is described in connection with the position of these yeast species in the subclass Heterobasidiomycetidae.     

Cultivation of human corneal endothelial cells isolated from paired donor corneas

Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability. Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage.The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.     

Microbial degradation of organophosphonates by soil bacteria

Bacteria that can utilize glyphosate (GP) or methylphosphonic acid (MPA) as a sole phosphorus source have been isolated from soil samples polluted with organophosphonates (OP). No matter which of these compounds was predominant in the native habitat of the strains, all of them utilized methylphosphonate. Some of the strains isolated from GP-polluted soil could utilize both phosphorus sources. Strains growing on glyphosate only were not isolated. The isolates retained high destructive activity after long-term storage of cells in lyophilized state, freezing to ?20°C, and maintenance on various media under mineral oil. When phosphorusstarved cells (with 2% phosphorus) were used as inoculum, the efficiency of OP biodegradation significantly increased (1.5-fold).     

Activation of nociceptin/orphanin FQ-NOP receptor system inhibits tyrosine hydroxylase phosphorylation, dopamine synthesis, and dopamine D(1) receptor signaling in rat nucleus accumbens and dorsal striatum

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D(1) receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D(1) receptor-stimulated cAMP formation, but failed to affect either adenosine A(2A) or DA D(2) receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D(1)-induced phosphorylation of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D(1) receptors, N/OFQ curtailed DA D(1) receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D(1) receptor signaling.