A high throughput method and culture medium for rapid screening of phosphate accumulating microorganisms

A novel PA Medium (PAM) for efficient screening of phosphate-accumulating organisms (PAOs) was developed taking Serratia marcescens NBRI1213 as model organism. The defined National Botanical Research Institute’s growth medium (NBRI) supplemented with 0.1% maltose, designed for quantitative estimation of phosphate accumulation was designated as PAM. Our work suggested usage of PAM for efficient qualitative screening and as a microbiological medium for preferential selection of PAOs on Petri-plates. For qualitative screening of PAOs, Toluidine blue-O dye (TBO) was supplemented in PAM, designated as PAM-TBO. Qualitative analysis of phosphate accumulated by various groups correlated well with grouping based upon quantitative analysis of PAOs, effect of carbon, nitrogen, salts, and phosphate accumulation-defective transposon mutants. For significantly increasing sample throughput, efficiency of screening PAOs was further enhanced by adaptation of PAM-TBO assay to microtiter plate based method. It is envisaged that usage of this medium will be salutary for quick screening of PAOs from environment.     

Effect of buffering on the phosphate-solubilizing ability of microorganisms

Native microflora present in the alkaline vertisols and two phosphate-solubilizing bacteria (PSB) isolated from soil using conventional screening media could not release phosphorus from alkaline Indian vertisol soils supplemented with carbon and nitrogen sources. The two PSBs could solubilize both rock phosphate and di-calcium phosphate in unbuffered media but failed to solubilize rock phosphate in buffered media. The organic acids secreted by these PSBs were 20–50 times less than that required to solubilize phosphorus from alkaline soil.     

印度洋可培养解有机磷细菌的多样性及解磷特性

【目的】筛选获得印度洋海水解有机磷细菌,研究其解有机磷作用机理,初步了解该地区可培养有机磷细菌的系统发育多样性。【方法】采用卵磷脂培养基从分离自印度洋的细菌中筛选解有机磷细菌,根据16S rRNA基因序列确定获得的有机磷细菌的分类地位;同时挑选解磷效果显著的3株细菌利用液体发酵进行产酶和解磷特性分析。【结果】自916株细菌中得到99株具有解有机磷活性的细菌,分属于16个属。在以卵磷脂为其有机磷来源时,菌株India-BSP-1 (Cobetia sp.)、India-BSP-21 (Pelagibaca sp.)、India-BSP-23 (Pelagibacterium sp.)的培养液中磷酸盐(DIP)浓度曲线为N型,并且碱性磷酸酶的检测活性滞后于DIP的生成。【结论】印度洋海水中解有机磷细菌种类多样性丰富,疑似有新种;解有机磷细菌的解磷特性受DIP和碱性磷酸酶相互作用的影响。     

杨树根际解无机磷细菌Mp1-Ha4的鉴定及其解磷机理

解无机磷细菌能够溶解土壤中的难溶性磷酸盐,增加土壤有效磷含量,促进植物生长。以一株杨树根际土壤中筛选得到的解无机磷细菌Mp1-Ha4为研究对象,利用分子生物学的方法对该菌株进行鉴定,测定了其对磷酸钙、磷酸铝和磷酸铁的解磷能力,并对该菌株9 d内的磷酸钙溶解动力学进行了研究。结果表明,解无机磷细菌Mp1-Ha4为一株西地西菌Cedecea sp.,其对磷酸钙的溶解能力远强于对磷酸铝和磷酸铁的溶解能力。在NBRIP液体培养基中,该菌株对磷酸钙的溶解能力达到了497.4 mg/L,在其对磷酸钙解磷过程中,培养基pH及可滴定酸度与解磷量分别呈显著负、正相关。高效液相色谱分析显示,该菌株在解磷过程中分泌了大量有机酸,主要包括α-酮戊二酸,酒石酸和苹果酸。分泌有机酸,降低环境pH可能是解无机磷细菌西地西菌(Cedecea sp.)Mp1-Ha4溶解难溶性磷酸盐的主要机制,同时该菌株对磷酸钙的高效溶解作用使其具有较大的研究和应用前景。     

入侵植物欧洲千里光内生固氮菌和溶磷菌多样性

【背景】菊科(Asteraceae)外来入侵植物欧洲千里光(Senecio vulgaris L.)来源于欧洲,广泛分布于我国西南和东北地区,在湖北高海拔山区也有分布。在入侵过程中,内生细菌可能在其获取氮磷营养方面起到了一些关键性作用。【目的】探究欧洲千里光内生固氮菌和溶磷菌的多样性和功能,为理解其入侵机制及防治提供参考。【方法】选择来自6个不同种群的种子,萌发后转移到花盆生长6-8周,并从每个种群中各挑选9株生长情况良好的植株,对其叶片和根组织表面进行消毒处理。使用基于nifH基因(固氮功能基因)的高通量测序方法对植物的固氮微生物群落结构和多样性进行研究。通过涂布平板法和平板划线法,在固体无氮培养基(Ashby)和无机磷培养基(inorganic phosphate, NBRIP)上对植物内生菌进行分离、纯化,对纯化的固氮菌株和溶磷菌株进行16S rRNA基因测序。采用钼锑抗比色法分析纯化溶磷菌株的溶磷能力。【结果】基于nifH基因的内生菌高通量测序结果表明,欧洲千里光叶样本中固氮菌多样性显著高于根样本;固氮菌群落中丰度最高的属是慢生根瘤菌属(Bradyrhizobium,30.9%...     

解磷菌PSB-R全基因组测序鉴定及其解磷特性分析

采用生理生化指标结合分子生物学技术对解磷菌PSB-R进行分类学鉴定,确定为粘质沙雷氏菌（Serratia marcescens）,通过二代测序平台Illumina NovaSeq PE150对PSB-R进行全基因组测序,分析预测了与解磷能力相关基因及其他植物促生基因组成情况。通过响应面优化试验检测了PSB-R最大解磷能力为805.199 mg/L,连续培养10代后解磷能力稳定且对多种难溶性磷酸盐均具有溶解能力。本研究为解磷菌解磷机制的进一步研究提供了基因组数据基础,同时证实PSB-R具有应用于菌肥的潜力,为后续解磷菌肥的研制提供了研究基础。     

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）。     

黄土高原天然和人工油松林根际土壤解磷细菌群落特征及其功能

为了揭示油松-根系微生物的互作关系及其对油松林分稳定性的影响,本研究选择陕西省黄龙县天然和人工油松林,采集油松根际与非根际土壤,测定非根际土壤化学特性,分离纯化根际土壤解磷(有机磷和无机磷)细菌,通过DNA基因测序鉴定解磷细菌,并测定解磷细菌的解磷能力。结果表明: 天然油松林非根际土壤中全碳(TC)、全氮(TN)含量以及C/N、N/P极显著高于人工油松林。2种油松根际土壤中共鉴定出20属65种解磷细菌,其中以芽孢杆菌属、链霉菌属和假单胞菌属为优势菌群;天然油松林根际解磷细菌多样性、丰富度和均匀度指数均高于人工油松林,而优势度指数低于人工油松林。链霉菌属与非根际土壤TC、TN和C/N、N/P呈正相关,而芽孢杆菌属和假单胞菌属与非根际土壤硝态氮、铵态氮、有效磷及全磷含量呈正相关。2种油松林根际土壤不同解磷细菌的解磷能力存在差异,其中天然和人工油松林根际共有的解磷细菌为假单胞菌Pseudomonas sp.34-5,其对磷酸钙的解磷能力最高,为11.40 μg·mL^-1;天然油松林根际独有的解磷细菌蕈状芽胞杆菌BF1-5对卵磷脂的解磷能力最高,为4.58 μg·mL^-1。该林区2种油松林根际解磷细菌菌群结构存在显著差异。与人工油松林相比,天然油松林根际土壤解磷细菌群落多样性更丰富且分布更均匀,解磷能力普遍高于人工林。     

Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation

In order to prepare the multi-functional biofertilizer, thermo-tolerant phosphate-solubilizing microbes including bacteria, actinomycetes, and fungi were isolated from different compost plants and biofertilizers. Except Streptomycesthermophilus J57 which lacked pectinase, all isolates possessed amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase activities. All isolates could solubilize calcium phosphate and Israel rock phosphate; various isolates could solubilize aluminum phosphate, iron phosphate, and hydroxyapatite. During composting, biofertilizers inoculated with the tested microbes had a significantly higher temperature, ash content, pH, total nitrogen, soluble phosphorus content, and germination rate than non-inoculated biofertilizer; total organic carbon and carbon-to-nitrogen ratio showed the opposite pattern. Adding these microbes can shorten the period of maturity, improve the quality, increase the soluble phosphorus content, and enhance the populations of phosphate-solubilizing and proteolytic microbes in biofertilizers. Therefore, inoculating thermo-tolerant phosphate-solubilizing microbes into agricultural and animal wastes represents a practical strategy for preparing multi-functional biofertilizer.     

铁尾矿区油松根际溶磷泛菌D2的筛选鉴定及溶磷特性

从河北省迁安市马兰庄镇铁尾矿植被恢复区油松根际分离出2株溶磷细菌,经过平板初筛和摇瓶复筛得到1株溶磷能力较强的菌株D2.通过菌落形态、生理生化特性及16S rDNA序列分析,确定此菌株D2属于泛菌属.利用液体发酵试验测定不同碳源、氮源对菌株D2溶磷能力的影响,通过高效液相色谱测定D2在不同氮源条件下产生有机酸的种类和浓度.结果表明:菌株D2对磷酸三钙有较强的溶磷能力,培养液有效磷含量最高为392.13 mg·L^-1,菌株D2的溶磷能力在碳源为葡萄糖、氮源为硫酸铵时效果最好;高效液相色谱测定发现,不同氮源条件下,D2分泌有机酸的种类和浓度存在差异,以硫酸铵、氯化铵、硝酸钾、硝酸钠、硝酸铵为氮源,均产生草酸、甲酸、乙酸和柠檬酸,以硫酸铵、氯化铵、硝酸铵为氮源还产生苹果酸.相关性分析表明,乙酸含量与有效磷含量间呈显著正相关(r=0.886,P<0.05),表明溶磷泛菌D2分泌的乙酸对无机磷的溶解有明显的促进作用,这也很可能是该菌株的重要溶磷机制之一.     

Comparison of several techniques for sampling macroinvertebrates in different habitats of a North Iberian pond

Several habitats of a Mediterranean pond were sampled for macroinvertebrates using different techniques: quantitative methods, semiquantitative hand-net sweep sampling and a qualitative protocol based on hand-net sweeps with fixed counts of individuals. The taxonomic composition was analyzed by DCA and several indices based on taxon composition or percentage of individuals were calculated. The aim of the study was to compare the results provided by these techniques and, in particular, to check if the qualitative approach could be an appropriate low-cost methodology for use in biomonitoring. The results provided by the three sampling methods in relation to submerged macrophytes were very similar. In soft sediment, certain differences were found between the quantitative and semiquantitative techniques. The qualitative protocol applied to single habitats collected fewer taxa than the corresponding quantitative or semiquantitative techniques. However, the overall protocol, consisting of samples from three habitats, provided higher richness values than all the quantitative and semiquantitative methods together, with 59 taxa out of the 70 collected during the study. It is concluded that this qualitative methodology performed reasonably well since it offers a reliable view of the community and it misses few taxa. Moreover, it is not so time consuming as other techniques and can be applied in a variety of habitats. It, therefore, seems to be an efficient method for bioassessment of the macroinvertebrate communities of ponds or the littoral zone of lakes.     

Reformulation of a new vancomycin analog: An example of the importance of buffer species and strength

The purpose of this research was to use our previously validated dynamic injection apparatus as a rapid method for screening pH-adjusted formulations of a new vancomycin analog, Van-An, for their potential to precipitate upon dilution. In 1 vial, Van-An was reconstituted according to the manufacturer’s instructions. In a separate vial, the Van-An formulation’s existing phosphate buffer species was supplemented with acetate buffer, which has a pKa in the desired range: between the pH values of the formulation (pH 3.9) and blood (pH 7.4). The formulations were injected using the dynamic injection apparatus into a flowing stream of isotonic Sorensen’s phosphate buffer at rates of 0.25, 0.5, 1, and 2 mL/min. The peaks obtained with the spectrophotometer were reproducible for each injection rate/formulation combination. For the phosphate-buffered formulation, the least amount of precipitation was obtained at the 0.25 mL/min injection rate. Acetate buffer was able to substantially reduce such precipitation, even at the highest injection rate. The opacity peaks for the formulation with the acetate addition were significantly smaller (P<.05) than those obtained for the unaltered formulation at all 4 injection rates. The results suggest that acetate is a better buffer species than phosphate for the pH range defined. Furthermore, we present evidence to support a generally applicable approach to screening new formulations of drug products that may be clinically useful for reducing the incidence of phlebitis in humans. Published: January 13, 2006     

Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides

Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species.     

Stabilization against hyperthermal denaturation through increased CG content can explain the discrepancy between whole genome and 16S rRNA analyses

Based largely upon analysis of ribosomal RNA, a third domain of life, called archaea, had been proposed in addition to bacteria and eukaryotes. However, quantitative analysis of 73 whole genomes shows only a two-domain division of life: into eukaryotes and prokaryotes. Thousands of orthologous genes in archaea and bacteria show an essentially unimodal distribution of sequence identities. Thus, whole genome analyses indicate that archaea are a phylum of bacteria rather than a separate domain of life. In contrast, archaeal rRNA and that of hyperthermophilic bacteria differ from the rRNA of mesophilic bacteria. Thus, there is a bimodal distribution of rRNA sequence identities which differ by 12%. This discrepancy in rRNA and gene content based analyses of whole genomes is likely due to a 15% elevated C:G content of the rRNA of archaea and hyperthermophilic bacteria. The elevated C:G content is consistent with stabilization against thermal denaturation caused by additional hydrogen bonding (3 bonds) in C:G pairs compared to A:U pairs (2 bonds). Based upon this premise, there is no reliable way to correct rRNA for such differences in base composition and it is not possible to quantitatively compare hyperthermophiles with mesophiles by the rRNA method. Furthermore, quantitative study of whole genomes shows that the extent of change in both bacterial and archaeal genes, including rRNA, has reached a limit. Thus, direct sequence comparisons work with closely related genomes, but it is not possible to differentiate the most divergent prokaryotic species, which are currently designated as separate phyla. We believe that the differences in characteristics of archaeal species is based primarily upon selection of genes and pathways compatible with the extreme environmental lifestyle, i.e., hyperthermophily.     

Phosphate solubilizing bacteria and their role in plant growth promotion

The use of phosphate solubilizing bacteria as inoculants simultaneously increases P uptake by the plant and crop yield. Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers. The principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil. Several phosphatase-encoding genes have been cloned and characterized and a few genes involved in mineral phosphate solubilization have been isolated. Therefore, genetic manipulation of phosphate-solubilizing bacteria to improve their ability to improve plant growth may include cloning genes involved in both mineral and organic phosphate solubilization, followed by their expression in selected rhizobacterial strains. Chromosomal insertion of these genes under appropriate promoters is an interesting approach.     

Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-amended clay, rice bran and rock phosphate-based granular formulation

Five phosphate-solubilizing bacteria (PSB) used in this study were isolated based on their ability to solubilize tricalcium phosphate (TCP) in Pikovskaya’s medium. Among the tested bacterial strains Burkholderia sp. strain CBPB-HIM showed the highest solubilization (363 μg of soluble P ml⁻¹) activity at 48 h of incubation. Further, this strain has been selected to assess its shelf life in nutrient-amended and -unamended clay, rice bran and rock phosphate (RP) pellet-based granular formulation. The results showed that the maximum viability of bacterium was observed in clay and rice bran (1:1) + 10% RP pellets than clay-RP pellets, irrespective of tested storage temperatures. Further, clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose supported the higher number of cells compared to glycerol-amended and nutrient-unamended pellets. In this carrier solubilization of Morocco rock phosphate (MRP) by Burkholderia sp. strain CBPB-HIM was also investigated. The maximum of water and bicarbonate extractable P (206 and 245 μg P g⁻¹ of pellet respectively) was recorded in clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose and glycerol respectively on day 5 of incubation. Therefore, this study proved the possibility of developing granular inoculant technology combining clay, rice bran and RP as substrates with phosphate-solubilizing Burkholderia.     

Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS/MS

In our study we have developed an HPLC-ESI-MS/MS method for qualitative and quantitative analysis of underivatized amino acids on dry blood spots. The sensitive and specific instrumental performances permitted the chromatographic separation of 40 amino acids and their isomers within 10 min. The method has been set up for cases of suspected metabolic diseases revealed by newborn screening. What is new is that it is applied on the same blood spots used for newborn screening, instead of plasma, in order to avoid involvement of doctors, increased anxiety for parents, stress for patients for plasma collection, long time of waiting and further costs for analysis.     

Plant Growth-Promoting Effect of the Chitosanolytic Phosphate-Solubilizing Bacterium Burkholderia gladioli MEL01 After Fermentation with Chitosan and Fertilization with Rock Phosphate

Phosphate-solubilizing bacteria (PSB) are important plant growth-promoting rhizobacteria that can increase soil fertility through the solubilization of insoluble inorganic phosphate and organophosphorus. In this study, a PSB, Burkholderia gladioli MEL01, was isolated and identified from rice–wheat rotation rhizosphere soil. MEL01 had an excellent phosphate-solubilizing capacity (reaching 107.69 mg/L) toward insoluble inorganic phosphate rock phosphate. HPLC analysis revealed that the mechanism of phosphate solubilization of MEL01 was probably due to secreted oxalic acid and gluconic acid transformation of phosphate from insoluble to soluble. MEL01 also exhibited 4030 U/L specific chitosanase activity when cultured with chitosan fermentation medium. Interestingly, the chitosan hydrolysis product chitooligosaccharide could significantly enhance the MEL01 phosphate-solubilizing capacity. Pot experiments showed that MEL01 chitosan medium fermentation liquor (MCMFL) could promote improvement of soil available phosphorus and pakchoi growth when supplemented with phosphate rock phosphate as the phosphate fertilizer. In addition, pot experiments demonstrated that MCMFL could also promote the growth of wheat, which could decrease the amount of compound fertilizer used. Microbial diversity analysis showed that the genera Pseudomonas, Burkholderia, Mycoplana, and Cellvibrio were enriched, which might participate in synergetic phosphate solubilization. Therefore, after fermentation with chitosan and fertilization with rock phosphates, MEL01 has potential as a phosphate biofertilizer in ecological agricultural production.

     

Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases

SUMMARY: A sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R(2) > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (<20%). The utility of our method is demonstrated with two types of applications: profiling of Calvin cycle intermediates in (i) dark-adapted and light-treated tobacco leaves, and in (ii) antisense plants expressing reduced levels of the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase or ribulose-1,5-bisphosphate carboxylase/oxygenase (comparison with wild-type controls). The broader applicability of our method is illustrated by profiling sugar phosphates extracted from the leaves of five taxonomically diverse plants.