Proteomic analysis of log to stationary growth phase Lactobacillus plantarum cells and a 2-DE database

Lactobacillus plantarum is part of the natural microbiota of many food fermentations as well as the human gastro-intestinal tract. The cytosolic fraction of the proteome of L. plantarum WCFS1, whose genome has been sequenced, was studied. 2-DE was used to investigate the proteins from the cytosolic fraction isolated from mid- and late-log, early- and late-stationary phase cells to generate reference maps of different growth conditions offering more knowledge of the metabolic behavior of this bacterium. From this fraction, a total of 200 protein spots were identified by MALDI-MS and a proteome production map was constructed to facilitate further studies such as detection of suitable biomarkers for specific growth conditions. More than half (57%) of the identified proteins were predicted to be involved in metabolic pathways of the bacterium. The protein profile changed during the growth of the bacteria such that 29% of the identified proteins involved in anabolic pathways were at least twofold up-regulated throughout the mid- and late-exponential and early-stationary phases. In the late-stationary phase, six proteins involved in stress or with a potential role for survival during starvation were up-regulated significantly.     

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.     

Improving fermentation consistency through better inoculum preparation

Substantial losses occur in the fermentation industry each year due to variability in yields and productivity. As the first stage in the process, inoculum consistency, in terms of size and quality, is clearly important. Yet, despite this, most inoculum development processes involve at least one highly variable transfer step, usually by wire loop, from a culture grown on a solid (agar) substrate. It is likely, then, that at least some of the variability in the production process can be attributed to a poorly controlled initial inoculation process.Experiments to determine the inherent variability of the conventional loop transfer technique showed a 12-fold variation in inoculum size. Although this can be improved by adopting a more rigid protocol, consistency is still poor. A simple alternative system, based on liquid transfers, leads to substantial improvements in the reproducibility of inoculum size and quality.C. Webb and S.P. Kamat are with the Department of Chemical Engineering, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK     

Dry-thermophilic anaerobic digestion of organic fraction of the municipal solid waste: focusing on the inoculum sources

The effect of inoculum source on anaerobic thermophilic digestion of separately collected organic fraction of municipal solid wastes (SC_OFMSW) has been studied. Performance of laboratory scale reactors (V: 1.1 L) were evaluated using six different inoculums sources: (1) corn silage (CS); (2) restaurant waste digested mixed with rice hulls (RH_OFMSW); (3) cattle excrement (CATTLE); (4) swine excrement (SWINE); (5) digested sludge (SLUDGE); and (6) SWINE mixed with SLUDGE (1:1) (SWINE/SLUDGE). The SC_OFMSW was separately and collected from university restaurant. The selected conditions were: 25% of inoculum, 30% of total solid and 55 degrees C of temperature, optimum in the thermophilic range. The six inoculum sources showed an initial start-up phase in the range between 2 and 4 days and the initial methane generation began over 10 days operational process. Results indicated that SLUDGE is the best inoculum source for anaerobic thermophilic digestion of the treatment of organic fraction of municipal solid waste at dry conditions (30%TS). Over 60 days operating period, it was confirmed that SLUDGE reactor can achieve 44.0%COD removal efficiency and 43.0%VS removal. In stabilization phase, SLUDGE reactor showed higher volumetric biogas generated of 78.9 mL/day (or 35.6 mLCH(4)/day) reaching a methane yield of 0.53 LCH(4)/gVS. Also, SWINE/SLUDGE and SWINE were good inoculums at these experimental conditions.     

Influence of inoculum age on hybridoma culture kinetics

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.     

Composting potential of different inoculum sources in the modified SEBAC system treatment of municipal solid wastes

The aim of the work described here was to analyse the biomethanization process for three types of organic fraction of municipal solid waste (OFMSW) using two different inoculum sources in a sequential leach-bed anaerobic reactor under thermophilic (55 degrees C) and dry conditions (20%TS). The OFMSWs studied were: separately collected food waste (SC_OFMSW) from restaurants, synthetic waste (ST_OFMSW) and mechanically selected municipal waste (MS_OFMSW). The inoculum studied were digested mesophilic sludge (SLUDGE) and digested SC_OFMSW. The results show that SLUDGE gave the best performance and this was therefore selected for the rest of the experiments. Three assays were performed to analyse the biomethanization processes of three types of organic waste. The results suggest that all three wastes give rise to an acclimation stage with acidogenic/acetogenic activity between days 5 and 20-30 and a stabilization phase associated with methanogenic activity. In conclusion, a mixture of OFMSW (regardless of its origin) and SWINE arranged in layers in the reactor, as well as SLUDGE used with an inoculum source, enhanced the fast start up phase of a modified sequential leach-bed system under dry thermophilic conditions.     

Profiling of Burkholderia cepacia secretome at mid-logarithmic and early-stationary phases of growth

Background

Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence.

Methodology/Principal Findings

In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence.

Conclusion/Significance

Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release.     

Influence of total solid and inoculum contents on performance of anaerobic reactors treating food waste

The aim of this paper was to analyze the biomethanization process of food waste (FW) from a university campus restaurant in six reactors with three different total solid percentages (20%, 25% and 30% TS) and two different inoculum percentages (20-30% of mesophilic sludge). The experimental procedure was programmed to select the initial performance parameters (total solid and inoculum contents) in a lab-reactor with V: 1100mL and, later, to validate the optimal parameters in a lab-scale batch reactor with V: 5000mL. The best performance for food waste biodegradation and methane generation was the reactor with 20% of total solid and 30% of inoculum: give rise to an acclimation stage with acidogenic/acetogenic activity between 20 and 60 days and methane yield of 0.49L CH4/g VS. Also, lab-scale batch reactor (V: 5000mL) exhibit the classical waste decomposition pattern and the process was completed with high values of methane yield (0.22L CH4/g VS). Finally, a protocol was proposed to enhance the start-up phase for dry thermophilic anaerobic digestion of food waste.     

裸脚菇0612-9液体菌种优化及其对后续发酵产活性物质的影响

裸脚菇0612-9次级代谢产物具有强烈抗青绿霉活性,可作为微生物源防腐剂用于柑橘保藏,但是其发酵周期长,产出能耗大效率低。用摇瓶对裸脚菇0612-9的液体菌种培养基、培养条件进行优化并对优化后液体菌种接种种龄、接种量进行探索,最后用5L发酵罐进行放大发酵验证。取样计数测定菌丝球数量、过滤称重测定菌丝干重、HPLC监测活性物质Ⅱ的积累、牛津杯法评价抗青绿霉活性。经研究最佳碳源为玉米粉和麦芽糖,最佳氮源为蛋白胨,最佳液体菌种培养基组成为：玉米粉30g/L、麦芽糖10g/L、蛋白胨15g/L、KH₂PO₄ 2g/L、MgSO₄·7H₂O 1g/L;最佳培养条件：起始pH 5、接种3×Ф7mm菌块、装液量100mL/250mL三角瓶、温度28℃、转速160r/min;优化前菌丝球数46个/10mL,菌丝干重0.28g/100mL,优化后菌球数达985个/10mL,菌丝干重达0.69g/100mL,分别为优化前的21.4倍、2.43倍;后续发酵使用种龄9d的液体菌种、接种量7.5%。优化后液体菌种在发酵罐中后续发酵周期从10d缩短至5d,缩短50%,产量比优化前提高8.28%。     

Influence of inoculum activity on the bio-methanization of a kitchen waste under different waste/inoculum ratios

The use of a granular inoculum prevented acidification during the anaerobic batch biodegradation of a kitchen waste for waste/inoculum ratios in the range of 0.5–2.3 g VS/g VS, when the alkalinity/COD ratio was 37 mg NaHCO₃/g COD. In similar experiments but using a suspended sludge with a significantly lower activity, the methane production rates and the biodegradability were significantly lower and the pH decreased below 5.5 at the waste/inoculum ratio of 2.3 g VS/g VS. When the added alkalinity was decreased to 2 mg NaHCO₃/g COD, the ratio waste/inoculum was clearly more important than the inoculum activity, since, irrespective of the sludge used, acidification occurred at waste/inoculum ratios higher than 0.5 g VS/g VS. The advantage of using granular sludge was further investigated in order to define reasonable condition of waste/inoculum ratio and added alkalinity that could be applied in practice. For a waste/inoculum ratio of 1.35, there were no significant differences between the results obtained for the biodegradability and maximum methane production rate (MMPR), when the alkalinity decreased from 44 to 22 mg NaHCO₃/g COD.     

Influence of inoculum size of Aspergillus parasiticus spores on aflatoxin production.

The influence of the inoculum size on growth and aflatoxin production was examined in Aspergillus parasiticus (NRRL 3145) by using a synthetic medium. The reduction in the number of spores by 4 to 5 log cycles either by serial dilution or by gamma irradiation caused a two fold increase in the toxin production. The decrease in the inoculum size induced a lag in growth of the culture, though the final yield of the mycelium over the 28-day experimental period was the same. The maximal accumulation of aflatoxin was observed on day 14 of incubation. A transition from the biphasic to monophasic pattern in aflatoxin production could be correlated with the size of the inoculum. The enhanced toxin production from dilute inocula was similar to that obtained with the surviving fraction of the spores after gamma irradiation (0 to 150 krads).     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

On-farm production of AM fungus inoculum in mixtures of compost and vermiculite

On-farm production of arbuscular mycorrhizal (AM) fungus inoculum can reduce the cost of the inoculum and increase utilization of this symbiosis in plant production. Bahiagrass (Paspalum notatum Flugge) seedlings, colonized by AM fungi, were transplanted into raised bed enclosures. Media within the enclosures was vermiculite mixed with either field soil or yard clippings compost in Experiment I and vermiculite mixed with yard clippings compost or dairy manure/leaf compost in Experiment II. Compost and vermiculite mixtures yielded more propagules of AM fungi than soil-based mixtures in Experiment I. Growth of plants in a 1:4 (v/v) mixture of yard clippings compost and vermiculite produced more inoculum (503 propagules cm(-3)) than growth in 1:9 and 1:99 (v/v) mixtures (240 and 42 propagules cm(-3), respectively). Water, inorganic nutrient solution minus P, and fish protein digest were added to inoculum production enclosures in Experiment II. Results indicated that supplemental nutrient addition was unnecessary. This method produces a concentrated inoculum of AM fungi in a form readily used as an amendment to horticultural potting media for the production of vegetable seedlings.     

Effects of rumen fluid collection site on microbial population structure during in vitro fermentation of the different substrates quantified by 16S rRNA hybridisation

Rumen fluid samples from a cow were withdrawn manually from the feed mat (solid phase) or the liquid phase below this mat and incubated in vitro with wheat straw, sorghum hay and a concentrate mixture. From the inoculum and several samples collected during in vitro incubation RNA was extracted to assess microbial population size and structure. RNA content recovered from the solid phase rumen fluid was significantly higher than from the liquid phase. The composition of the microbial population in the solid phase material was characterised by a high proportion of Ruminococci. Neither the proportion of other cell wall degrading organisms (Fibrobacter and Chytridiomycetes) nor the Eukarya and Archaea populations differed between the two sampling sites. Gas production was higher when substrates were incubated with solid phase than with liquid phase rumen fluid regardless of sampling time. However, the higher level of gas production was not accompanied by a corresponding increase in true digestibility. The RNA probes showed that during in vitro incubation with liquid phase rumen fluid, the eukaryotic population was inactive no matter which substrate was used and the activity of methanogens (Archaea) was lower than with solid phase rumen fluid. The population pattern of the cell wall degrading organisms was influenced mainly by the substrate fermented, and to a smaller extent by the inoculum used for in vitro fermentation.     

Statistical design of the effect of inoculum conditions on growth of hairy root cultures of Catharanthus roseus

Summary The factors in the inoculum conditions that influence the growth rate of hairy roots of Catharanthus roseus in liquid culture were investigated in a randomized two-level, three-factor statistical design. The three variables were the number of root tips, the length of root tips, and the initial volume of media. The experiments and analysis demonstrated the effect of inoculum conditions on hairy root growth, with the length of hairy root tips being the dominant variable without any clonal variability. The best growth rates (doubling times) were obtained with an inoculum of 5 hairy root tips, each 35–40 mm long in 50 mL media: 3.7 and 3.1 days for C. roseus hairy root clones LBE-6-1 and LBE-4-2, respectively.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848     

Variability in maturation and germination from white spruce somatic embryos,as affected by age and use of solid or liquid culture

Summary The yield of morphologically normal Stage 3 somatic embryos of white spruce [Picea glauca (Moench) Voss], and subsequent germinability, was affected by culture age and use of solid and/or liquid culture growth conditions. Of the conditions that were compared, best results were obtained with cultures up to 3 yr old that had been continuously grown in liquid medium. Such material yielded up to 374 morphologically normal Stage 3 embryos per g f. wt. inoculum, when routinely pretreated using a 1 wk 2,4-dichlorophenoxyacetic acid-free period before maturation. By comparison the continual use of solid culture conditions resulted in lower yields (5/g f. wt. inoculum), and the use of solid medium in combination with liquid medium showed a greater affect of age on the production of normal Stage 3 embryos (348/g f. wt at 1.5 yr down to 19/g f. wt. at 3 yr) over the age range tested. In the absence of culture pretreatment, the oldest liquid cultures yielded only 44 normal Stage 3 embryos/g f. wt. inoculum, and the comparable solid to liquid cultures yielded 1.3/g f. wt. inoculum. The number of aberrant Stage 3 embryos in older cultures was reduced as a result of culture pretreatment; for example, in the oldest liquid cultures these represented 83% of the Stage 3 embryo population without pretreatment and 45% with pretreatment. Normal Stage 3 somatic embryo yield and germination characteristics (radicle and epicotyl development) were informative in distinguishing among the conditions studied. Germination characteristics were especially important when maturation responses were incapable of distinguishing among age classes. NRCC Contribution no. 38462.     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.     

Effects of sucrose, inoculum density, auxins, and aeration volume on ceil growth ofGymnema sylvestre

To improve the cell protocol forCymnema sylvestre, we investigated the influence of initial sucrose concentration, inoculum density, and optimal concentrations of auxins (IBA and NAA) in flask cultures, as well as the role of aeration volume in bioreactor cultures. Cell growth was enhanced 9-fold when the medium was supplemented with 3% sucrose versus a sucrose-free environment. Increasing the inoculum density to 60 g (wet weight) L^-1, but no further, greatly improved the growth of these cultures. All concentrations of IBA proved inhibitory while supplementation with 5 nig L^-1 NAA was associated with significantly higher dry-cell weights. In our bioreactor cultures, a step-wise increase in aeration volume from 0.05 to 0.40 wm was optimal for cell growth. Although biomass (i.e., fresh weight) accumulated in the bioreactor up until Day 20, the dry-cell weights increased 10-fold, but only through Day 15. The internal dynamics of our culture media indicated that sucrose was preferentially utilized and that its concentration steeply decreased at the log phase. In contrast, both glucose and fructose supplies were exhausted only at the beginning of the declining phase. Our findings suggest that a 15-d culture period is optimal for G.sylvestre cell growth in a bioreactor.