Comparison of treatments for the in vitro detachment of cellulolytic bacteria from ruminal digesta samples

Four treatments were applied to ruminal digesta samples in a randomized complete block design experiment to evaluate their effect on the extent of detachment of ruminal cellulolytic bacteria. The treatments were: blending with anaerobic dilution solution (ADS), blending with ADS after a 6-h cold incubation, blending with ADS+Tween 80, and blending with ADS +Tween 80 after a 6-h cold incubation. Compared with blending in ADs alone, significant increases (p<0.2) of 10% and 12% in the number of cellulolytic bacteria were observed with the 6-h cold and the 6-h cold plus Tween 80 treatments, respectively. Tween 80 had no effect on the number of cellulolytic bacteria released from ruminal digesta samples irrespective of chilling. It was concluded that these additional treatments afforded little value over blending in buffer alone in detaching cellulolytic bacteria from ruminal digesta.     

Effects of Prechilling and Sequential Washing on Enumeration of Microorganisms from Refuse

Techniques were evaluated for formation of a liquid inoculum from shredded municipal refuse, including chilling the refuse at 4°C prior to blending and multiple washing and blending cycles. The average count of cellulolytic bacteria from six different detachment treatments was 5.1 × 10⁴ cells per g (dry weight) of refuse with a range of 0.7 × 10⁴ to 12.7 × 10⁴ cells per g (dry weight). The liquid obtained from blending the refuse in phosphate buffer followed by hand squeezing was the selected detachment procedure. The inoculum formation procedure was validated by the addition of ruminal cellulolytic bacteria to refuse and recovery of the cellulolytic bacteria by most-probable-number enumerations. The ratio of measured to expected cell counts among tests in which different volumes of ruminal fluid were added to refuse ranged from 2.7 to 14.4. There was no evidence of anaerobic cellulolytic fungi in a refuse sample.     

Electron microscopic study of the methylcellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers

The presence of methylcellulose prevents the attachment of cellulolytic rumen bacteria to cellulose fibers. The addition of methylcellulose to pure cultures of these organisms in which the cells are already adherent to cellulose causes their detachment from this insoluble substrate and the inhibition of their growth. Methylcellulose is not used as a carbon source by these organisms and has no effect on their growth when glucose and cellobiose are the carbon sources. Attached cells of Bacteroides succinogenes orient themselves in the plane of the individual cellulose fibers and their methylcellulose-induced detachment, which is complete (almost 100%), leaves grooves where the cellulose has been digested. Attached cells of Ruminococcus albus colonize the cellulose in a looser and less regular pattern and their almost complete methylcellulose-induced detachment leaves less regular pits in the cellulose surface. On the other hand, attached cells of Ruminococcus flavefaciens colonize the cellulose surface in a random orientation by means of a discernible exopolysaccharide network, and their less complete methylcellulose-induced detachment leaves no residual impressions on the cellulose surface. These data support the suggestion that bacterial attachment is necessary for the digestion of highly ordered crystalline cellulose, and that cellulolytic species differ in the nature of their attachment to this insoluble substrate and in the nature of their enzymatic attack. Methylcellulose is an effective agent for detaching major rumen cellulolytic bacteria from their cellulosic substrate.     

Response of Solenopsis invicta (Hymenoptera: Formicidae) to potassium oleate water solution

Abstract In contrast to floating on the surface of distilled water, ants were immediately submerged after being placed in the potassium oleate (PO) water solution, which led to immobilization within minutes. However, some workers survived after being immersed in 0.03% PO water solution at 25°C for up to 640 min. Elevated temperature of the PO water solution is needed to kill ants within a shorter time frame. At 50°C and 0.13% PO concentration, total mortality was achieved with 10-min immersion for all ants, including brood and adult ants. Soil has a negative effect on the effectiveness of potassium oleate; however, such negative effect can be overcome by increasing either treatment temperature or duration of the treatment. In addition to immobilizing and lethal effect, PO repels ants. PO may have the potential to be incorporated into immersion treatment for the quarantine of imported fire ants to reduce the use of synthetic contact insecticides.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

On the surface properties of oleate micelles and oleic acid/oleate vesicles studied by spin labeling

Dilute aqueous systems composed of sodium oleate micelles and sodium oleate/oleic acid vesicles were investigated as a function of pH by electron spin resonance spectroscopy with TEMPO-stearate TEMPO-stearamide as well as with a positively charged water soluble spin label, TEMPO-choline. The dynamics of the three TEMPO-spin labels were found to be sensitive to changes in the interfacial region of the aggregates as a function of pH. The results obtained are consistent with the formation of a hydrogen bond network (RCOO⁻ ↔ HOOCR) at the surface of the sodium oleate/oleic acid system in the course of the transformation of micelles into the closed bilayers (vesicles). Vesicles formation below pH 10 was determined independently with a spin labeled glucose derivative.     

Alteration of the growth rate and lag time of Leuconostoc mesenteroides NRRL-B523.

Bacterial profile modification is an important enhanced oil recovery technique used to direct injected water into a reservoir's low permeability zone containing trapped crude oil. During water flooding, the use of bacteria to plug the high permeability water zone and divert flow into the oil-bearing low-permeability zone will have a significant economic impact. However, during the field implementation of bacterial profile modification, the rapid growth of bacteria near the injection well bore may hinder the subsequent injection of growth media so that profile modification of the reservoir occurs only in the immediate vicinity of the well bore. By slowing the growth rate and prolonging the lag phase, the onset of pore-space plugging may be delayed and the biologically active zone extended deep into the reservoir. High substrate loading, high pH values, and the addition of the growth inhibitors sodium dodecylsulfate and sodium benzoate have been used in combination to alter the growth characteristics of Leuconostoc mesenteroides NRRL-B523 grown in batch conditions. The highest sucrose concentration used in these studies, 500 g/L, produced lag times 12-fold greater than the slowest lag times achieved at low sucrose concentrations. When L. mesenteroides was grown in media containing 500 g/L sucrose, an alkaline pH value threshold was found above which bacteria did not grow. At this threshold pH value of 8.1, an average lag time of 200 h was observed. Increasing the concentration of sodium benzoate had no effect on lag time, but reduced the growth rate until the threshold concentration of 0.6%, above which bacteria did not grow. Last, it was found that a solution of 0.075 mM sodium dodecylsulfate in media containing 15 g/L sucrose completely inhibited bacterial growth.     

The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor

In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.     

Effect of caries preventive products on the growth of bacterial biofilm on titanium surface

Fluorides may affect the oxide layer on titanium surface. Caries preventive mouthwashes or gels contain fluorides and are applied at low pH. The aim of the present work was to study whether various concentrations of fluoride at acidic pH cause changes in the surface structure on the polished region of Ti implants, and alter the adherence and colonization of bacteria. Commercially pure Ti grade 4 discs with a polished surface were treated with a mouthwash containing 0.025% fluoride, a gel containing 1.25% fluoride or a 1% aqueous solution of NaF (pH 4.5). The change of surface roughness of the samples and the colonization of Porphyromonas gingivalis strains were studied by scanning electron microscopy after 5 days of anaerobic incubation. The quantity of the bacterial protein was determined by protein assay analysis. Agents with high fluoride concentration at acidic pH increased the roughness of the Ti surface. A slight increase in the amount of bacteria was found on the surfaces treated with 1% NaF and gel in comparison with the control surface. This study suggested that a high fluoride concentration at acidic pH may hinder the development of a healthy transgingival epithelial junction on Ti implants, due to bacterial colonization.     

Impact of barley form on equine total tract fibre digestibility and colonic microbiota

This study aimed at assessing the impact of four barley forms on total tract apparent digestibility of dietary fibre in horses fed a large amount of starch in the morning meal (0.27% BW). Processed barley forms had a greater pre-caecal starch digestibility than the whole form. Based on this result, we hypothesised that using barley-processing methods would limit the potential dumping of undegraded starch in the hindgut of horses and, consequently, the potential negative effect on fibre degradation in the hindgut. In a 4×4 latin square design, four mature geldings fitted with a right ventral colon-fistula were fed a meadow hay : concentrate (62 : 38; dry matter (DM) basis) diet at 1.7% BW. The concentrate was made of 80% barley distributed either as whole grain or as processed forms: 2.5 mm ground, pelleted or steam-flaked. For each period, total tract apparent digestibilities of DM, NDF and ADF were determined over 3 consecutive days by total faecal collection, whereas pH, volatile fatty acids (VFA) concentrations and cultural functional bacteria counts (total anaerobic, cellulolytic bacteria, lactic acid producers, amylolytic bacteria and lactic acid utilisers) in colonic content were evaluated on 1 day 4 h after the morning meal. Total tract apparent digestibility of DM and dietary fibre was influenced (P<0.05) by barley form. Diets including thermo-mechanically treated barley forms led to a higher (P<0.05) total tract apparent digestibility of NDF than those constituted of ground barley and also led to a greater (P<0.05) total tract apparent digestibility of ADF than those made of whole or ground barley forms. However, no significant difference was observed in colonic pH, VFA concentrations and cultural bacteria concentrations. Owing to a high starch supply in the morning meal, the concentration of the functional bacteria in the colonic content averaged 7.8 log CFU/ml, 5.9 NPM/ml, 6.9 and 7.3 CFU/ml for total anaerobic, cellulolytic, amylolytic and lactic acid-utilising bacteria, respectively. Consequently, providing horses with pelleted or steam-flaked instead of ground barley forms may limit the negative impact of starch on fibre digestibility in horses fed a high level of starch in the morning meal (0.27% BW). Moreover, the fibre-to-starch ratio fed in this experiment did not cause any digestive upset.     

利用定量杂交法检测绵羊瘤胃纤维细菌的研究

实验利用通用细菌探针和3株纤维分解菌的特异性探针,初步建立起对瘤胃细菌进行检测的16SrRNA定量杂交的方法。试验将提取的总RNA按浓度系列稀释后与通用细菌探针进行杂交,检测结果所做的回归分析表明,杂交信号与尼龙膜上的所点RNA的量具有明显线性关系。同时对几份瘤胃样品进行3种纤维分解菌的初步定量检测,结果显示3种纤维分解菌的相对丰度与前人报道相似,表明该方法能够对瘤胃细菌进行定量分析,可在后续相关研究中使用。     

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.     

Rumen microbial changes in cattle fed diets with or without salinomycin

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Rumen microbial changes in cattle fed diets with or without salinomycin.

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Recovery and quantification of bacterial cells associated with streambed sediments

Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the (3)H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.     

Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium.

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

Enumeration and activity of cellulolytic bacteria from gestating swine fed various levels of dietary fiber.

Cellulolytic bacteria were enumerated and cellulase activity was determined over a 98-day period from fecal samples of gestating swine fed various levels and sources of fiber. The diets, each fed to five pigs, were a corn-soybean control, 20% corn cobs, and 40 and 96% alfalfa meal. Fecal samples were collected from all pigs on days 0, 5, 14, 21, 35, 49, 70, and 98. Overall, the most probable number of cellulolytic bacteria from pigs fed the control, 20% corn cobs, and 40 and 96% alfalfa meal was 23.3 X 10(8), 15.2 X 10(8), 45.1 X 10(8), and 52.5 X 10(8) per g (dry weight) of fecal sample, respectively. Enumeration of cellulolytic bacteria by counting zones of clearing in roll tubes, as compared with the most probable number procedure, accounted for only 1.1 and 17.0% of the cellulolytic bacteria, respectively, from pigs fed the control or 96% alfalfa meal diet. Cellulolytic bacteria (most probable number) on days 70 and 98 accounted for 4.1 and 10.0% of the viable count for the pigs fed the control and 96% alfalfa meal diets, respectively. The viable count was not different between pigs fed the control and 96% alfalfa meal diets. The overall mean cellulolytic activity (milligrams of glucose released from carboxymethyl cellulose per gram [dry weight] fecal sample per 30 min was 17.0, 19.9, 23.8, and 20.6, respectively, for the control, 20% corn cobs, and 40 and 96% alfalfa meal diets. The results indicate that the cellulolytic flora can be increased by prolonged feeding of high-fiber diets and may represent 10% of the culturable flora.     

Microbial production of biogas form organic wastes

Biogas production through bacterial digestion of water hyacinth, rice straw and sawdust inoculated with cow dung was investigated in a batch loading of laboratory digestors. It was found that the overall gas yield was 368.00 l from water hyacinth having 77.13% methane, 310.00 l from rice straw with 77.80% methane, and 144.80 l from sandust containing 62.28% methane. Along with the production of gas, the change in pH and microbial population were also monitored. The pH of the digestion mixture was initially low and found to be increasing as the digestion progressed. The total microbial population of bacteria, fungi, actinomycetes, cellulolytic organisms, and coliform bacteria were found to decrease with time.     

Enumeration and activity of cellulolytic bacteria from gestating swine fed various levels of dietary fiber

Cellulolytic bacteria were enumerated and cellulase activity was determined over a 98-day period from fecal samples of gestating swine fed various levels and sources of fiber. The diets, each fed to five pigs, were a corn-soybean control, 20% corn cobs, and 40 and 96% alfalfa meal. Fecal samples were collected from all pigs on days 0, 5, 14, 21, 35, 49, 70, and 98. Overall, the most probable number of cellulolytic bacteria from pigs fed the control, 20% corn cobs, and 40 and 96% alfalfa meal was 23.3 X 10(8), 15.2 X 10(8), 45.1 X 10(8), and 52.5 X 10(8) per g (dry weight) of fecal sample, respectively. Enumeration of cellulolytic bacteria by counting zones of clearing in roll tubes, as compared with the most probable number procedure, accounted for only 1.1 and 17.0% of the cellulolytic bacteria, respectively, from pigs fed the control or 96% alfalfa meal diet. Cellulolytic bacteria (most probable number) on days 70 and 98 accounted for 4.1 and 10.0% of the viable count for the pigs fed the control and 96% alfalfa meal diets, respectively. The viable count was not different between pigs fed the control and 96% alfalfa meal diets. The overall mean cellulolytic activity (milligrams of glucose released from carboxymethyl cellulose per gram [dry weight] fecal sample per 30 min was 17.0, 19.9, 23.8, and 20.6, respectively, for the control, 20% corn cobs, and 40 and 96% alfalfa meal diets. The results indicate that the cellulolytic flora can be increased by prolonged feeding of high-fiber diets and may represent 10% of the culturable flora.