Response of selected poultry cecal probiotic bacteria and a primary poultry salmonella typhimurium isolate grown with or without glucose in liquid batch culture

Abstract

The objective of this study was to determine whether fermentation by a cecal probiotic co‐culture of an Enterococcus sp. and Veillonella sp. would inhibit the in vitro growth of a S typhimurium poultry isolate. The growth rates of S. typhimurium and Enterococcus were significantly reduced at pH 5. At the two pH levels, there was a significant (p < 0.001) increase at 24 h in colony forming units for each of the bacteria enumerated from the mixed culture compared to the respective pure culture enumerations. S. typhimurium was not inhibited in mixed cultures. The mixed cultures produced more acetate than any of the pure cultures and lactate produced by Enterococcus appeared to be utilized by Veillonella.     

Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella typhimurium

This study demonstrates that the growth of S. typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell-association and the ability to invade cultured HEp-2 cells. Cell-association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7. The results suggest that the growth rate of the culture may have affected cell-association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell-association and invasion. However, the results also suggest that the individual SCFA may play a role in modulating cell-association and the invasion phenotype and the regulation of cell-association and invasion by the SCFA was dependent on the concentration and the pH of the medium. Although the growth rates were similar for S. typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell-association and invasion were observed among these cultures. Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar.     

The combination of zinc compounds and acidic pH limits aerobic growth of a Salmonella typhimurium poultry marker strain in rich and minimal media

The objective of the present study was to examine the combined effects of zinc compounds with different acidic pH levels on the aerobic growth of a S. typhimurium poultry isolate in either rich or minimal media. When overall main effects of pH levels of medium or concentrations of Zn compounds were compared, growth rates of the S. typhimurium poultry isolate were significantly (p < 0.05) decreased by stepwise increase of pH levels of medium (pH 4, 5, 6, and 7) or concentrations (0.67, 3.35, and 6.03%) of Zn compounds (Zn acetate and Zn sulfate). In general growth rates of S. typhimurium poultry isolate appeared to be more reduced by Zn acetate than by Zn sulfate and more reduced in minimal media compared to rich media.     

Isolation and physiological characterization of the pentachlorophenol degrading bacterium Sphingomonas chlorophenolica

Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant that has been used widely as a general biocide in commercial wood treatment. Owing to the rapid industrial growth, serious soil and water pollutions by chlorophenols has been reported in Taiwan. In this study, 10 indigenous PCP-degrading bacterial strains were isolated from a PCP-degrading mixed culture, and the potential of both the pure and mixed cultures for PCP degradation compared. Moreover, the physiological characteristics and optimum growth conditions of the PCP-degrading bacteria were investigated. One of the isolated bacterial strains with good potential for PCP degradation was characterized and identified as Sphingomonas chlorophenolica by 16S rDNA gene analysis. The result of the optimum growth temperatures revealed that this organism was a mesophile. The optimum pH for PCP removal by S. chlorophenolica was between 6.9 and 7.6. Increase in concentration of PCP has a negative effect on the biodegradation potential of S. chlorophenolica and PCP concentration above 600 mg l(-1) was inhibitory to its growth. The results of this study indicate that this S. chlorophenolica strain has a better potential for PCP degradation compared to the enriched mixed culture. The physiological characterization of the isolates also indicates the possible application of this strain for bioremediation of sites contaminated with PCP.     

Microbial and kinetic characterization of pure and mixed cultures aerobically degrading 4-nitrophenol

The molecular and kinetic characterization of a microorganism able to aerobically degrade 4-nitrophenol (4NP) is presented. The microorganism was isolated from a mixed culture operating in a laboratory-scale sequencing batch reactor with an aerobic anoxic cycle. It was identified as a member of Ralstonia genus within Betaproteobacteria. It is a gram negative coccobacillum (cell length of 2-3 microm) able to aerobically store lipid inclusions when grown aerobically on nitrophenol as the sole carbon source in the range of tested concentrations (80-320 mg l(-1)). Batch kinetic tests were performed with the pure culture, while the kinetics of the mixed biomass was directly investigated in the reactor. For pure cultures exponential growth was observed, with growth rate values in the range of 2-6 d(-1); in experiments with the mixed cultures 4NP concentrations were correlated with growth using the Haldane equation (k(max) = 0.30 mg 4NP mg(-1) VSSh(-1); K(s) = 55 mg 4NPl(-1) and K(I) = 15 mg 4NPl(-1)). Observed pure culture growth rates were higher than those of mixed cultures. This result can be explained by considering that in mixed culture the biomass is evaluated as volatile suspended solids, including both specialized biomass for 4NP removal and denitrifying bacteria.     

不适宜生长条件对混茵降解苯胺的影响

分别从台州和衢州某化工厂的好氧池中分离筛选得到2株苯胺降解菌TZl和JH1,经16SrDNA测序鉴定为Comamonassp．TZ1和Pseudomonassp．JH1,均具有较强的苯胺降解能力,培养24h后,可使初始浓度为800mg／L的苯胺去除率达到96．4％～98．4％。在此基础上,按体积比l：1将2株菌液进行混合构建了混合菌体系,进而对比考察了苯胺初始浓度、pH、盐度和重金属等环境因子对单一菌和混合菌生长量及降解苯胺效果的影响,重点探讨混合菌对不适宜生长环境的适应性及其对苯胺的降解特性。通过单一菌和混合菌对比实验发现,在适宜苯胺初始浓度、pH和盐度条件下,混合菌的生长量略高于单一菌;在不适宜生长的高浓度苯胺、pH和盐度条件下,混合菌也表现出了更强的适应性和苯胺矿化能力。Zn2＋和Cr6＋耐受实验则表明,对于Cr6＋混合菌表现出了更强的耐受能力,而对于zn2＋并没有表现出更强的耐受能力。     

不适宜生长条件对混菌降解苯胺的影响

分别从台州和衢州某化工厂的好氧池中分离筛选得到2株苯胺降解菌TZ1和JH1,经16S rDNA测序鉴定为Comamonas sp.TZ1和Pseudomonas sp.JH1,均具有较强的苯胺降解能力,培养24 h后,可使初始浓度为800 mg/L的苯胺去除率达到96.4%~98.4%。在此基础上,按体积比1∶1将2株菌液进行混合构建了混合菌体系,进而对比考察了苯胺初始浓度、pH、盐度和重金属等环境因子对单一菌和混合菌生长量及降解苯胺效果的影响,重点探讨混合菌对不适宜生长环境的适应性及其对苯胺的降解特性。通过单一菌和混合菌对比实验发现,在适宜苯胺初始浓度、pH和盐度条件下,混合菌的生长量略高于单一菌;在不适宜生长的高浓度苯胺、pH和盐度条件下,混合菌也表现出了更强的适应性和苯胺矿化能力。Zn2+和Cr6+耐受实验则表明,对于Cr6+,混合菌表现出了更强的耐受能力,而对于Zn2+并没有表现出更强的耐受能力。     

Sodium bisulfate and a sodium bisulfate/tannin mixture decreases pH when added to an in vitro incubated poultry cecal or fecal contents while reducing Salmonella Typhimurium marker strain survival and altering the microbiome

The objective of the present study was to investigate the ability of animal feed-grade sodium bisulfate (SBS) and a mixture of sodium bisulfate/tannin to inhibit the growth of Salmonella using an anerobic in vitro mixed cecal culture to mimic the conditions within the chicken cecum. An initial inoculum of Salmonella Typhimurium was introduced to an anerobic dilution solution containing 1/3000 diluted cecal bacteria and solids consisting of ground chicken feed and different percentages of solid SBS or SBS/tannin, and surviving organisms were enumerated. Two different experimental designs were employed. In the “unadapted” treatment, the S. Typhimurium was added at the beginning of the culture incubation along with cecal bacteria and chicken feed/SBS or chicken feed/SBS/tannin. In the “adapted” treatment, S. Typhimurium was added after a 24 hour pre-incubation of the cecal bacteria with the chicken feed/SBS or chicken feed/SBS/tannin. Adding SBS resulted in reduction of pH in the cultures which paralleled with the reduction of S. Typhimurium. The SBS alone was found to be inhibitory to S. Typhimurium in the adapted treatment at all concentrations tested (0.25, 0.5, and 0.75%), and the degree of inhibition was concentration-dependent. Salmonella Typhimurium was completely killed in the adapted culture with 0.5% SBS after 24 and 48 h. The SBS/tannin mixture was less inhibitory than SBS alone at the same concentrations in side-by-side comparisons. Testing at a 0.5% SBS concentration, chicken age had little or no effect on log reduction of S. Typhimurium relative to age-matched control cultures without SBS, but age did affect the absolute number of S. Typhimurium surviving, with the greatest decreases occurring at 2 and 4 weeks of age (approx. 10³ S. Typhimurium surviving) compared to 6 weeks of age (approx. 10⁵ Salmonella surviving). Microbiome analysis with an Illumina MiSeq platform was conducted to investigate bacterial compositional changes related to the addition of SBS. The relative abundance of Firmicutes (at the phylum level) was decreased, and genera Lactobacillus and Faecalibacterium were increased when SBS was added to the anaerobic mixed culture containing either fecal or cecal material. The antimicrobial action of feed-grade SBS may represent a potential pre-harvest control measure for Salmonella in poultry production.     

Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste

Eight aerobic bacterial strains were isolated from pulp paper mill effluent sludge. Out of eight through nutrient enrichment technique three potential aerobic bacterial strains ITRC S(6), ITRC S(7) and ITRC S(8) were found capable to effectively degrade the kraft lignin (KL), a major byproduct of the chemical pulping process and main contributor to the colour and toxicity of effluent. Further, these potential strains (ITRC S(6), ITRC S(7) and ITRC S(8)) were biochemically characterised as Gram variable small rod, Gram negative rod and Gram positive rod respectively. Subsequently, 16S rRNA sequencing showed 95% base sequence homology and it was identified as Paenibacillus sp. (AY952466), Aneurinibacillus aneurinilyticus (AY856831), Bacillus sp. (AY952465) for ITRC S(6), IITRC S(7) and ITRC S(8), respectively. In batch decolourization experiments Bacillus sp. ITRC S(8) reduced the colour of lignin amended mineral salt medium, pH 7.6 by 65% after 6th d, at 30 degrees C, A. aneurinilyticus ITRC S(7) by 56% and Paenibacillus ITRC S(6) 43%. Under these conditions the three strains degraded the KL by 37%, 33% and 30%, respectively while the mixed culture of these three bacteria reduced colour by 69%, lignin by 40% and total substrate by 50% under same conditions. Biodegradation of the KL was not affected by low (<0.2 mg l(-1)) dissolved oxygen content; thus oxygen inhibition is more likely to be a metabolism-dependent event. Initially with 48 h incubation the decolourization was slow with decreased pH. Further incubation there was rapid decolourization with slight increase in pH at 6d compared with initial pH by increasing culture optical density. The lignin analysis from medium with HPLC indicated complete degradation rather than biotransformation with complete loss of absorbance peak at 280 nm.     

The impact of aluminium on green algae isolated from two hydrochemically different headwater streams, Bavaria, Germany

Two strains of green algae (Scenedesmus sp. and Chlorella sp.) have been isolated from two headwater streams differing in stream chemistry (Eger river: acidic, low alkalinity; Püttlach river: slightly alkaline, high alkalinity). In this study the growth response of these indigenous algae to increased concentrations of aluminium (Al) is investigated. A semi-continuous culture technique was used for Al-toxicity studies. Algal response was determined by calculating growth rates from turbidity and cell counts. Those Al-species which are well known to be toxic were estimated by equilibrium calculations using the WATEQ computer program (Truesdall & Jones, 1973). The pH-value of the culture media was usually pH=5, except for one of the test series. Tested concentrations ranged from c=4 to 220 micromol litre(-1). The isolated strains of green algae were highly sensitive to increased Al-concentrations. The strain isolated from the Püttlach river was more sensitive than the Eger river algae. A total growth inhibition occurs at Al-concentrations of c=4 micromol litre(-1) if the whole Al was added at once. If Al was added gradually into the growth media the response of the algae was delayed. This is due to Al-enrichment in cells. In our long term toxicity studies, growth inhibition occurs even at nearly neutral pH-conditions (pH=6.5) although Al toxicity is expected at pH-values less than pH=5.5. This new result confirms the need of long-term studies in continuous cultures under simulated natural conditions. This might be the only way to achieve valid conclusions about the fate and the toxicity of environmental pollutants.     

Enrichment and isolation of a mixed bacterial culture for complete mineralization of endosulfan

In the present study, we isolated three novel bacterial species, namely, Staphylococcus sp., Bacillus circulans-I, and Bacillus circulans-II, from contaminated soil collected from the premises of a pesticide manufacturing industry. Batch experiments were conducted using both mixed and pure cultures to assess their potential for the degradation of aqueous endosulfan in aerobic and facultative anaerobic condition. The influence of supplementary carbon (dextrose) source on endosulfan degradation was also examined. After four weeks of incubation, mixed bacterial culture was able to degrade 71.82 +/- 0.2% and 76.04 +/- 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively, with an initial endosulfan concentration of 50 mg l(-1). Addition of dextrose to the system amplified the endosulfan degradation efficiency by 13.36 +/- 0.6% in aerobic system and 12.33 +/- 0.6% in facultative anaerobic system. Pure culture studies were carried out to quantify the degradation potential of these individual species. Among the three species, Staphylococcus sp. utilized more beta endosulfan compared to alpha endosulfan in facultative anaerobic system, whereas Bacillus circulans-I and Bacillus circulans-II utilized more alpha endosulfan compared to beta endosulfan in aerobic system. In any of these degradation studies no known intermediate metabolites of endosulfan were observed.     

Metabolism of chlorinated biphenyls: use of 3,3'- and 3,5-dichlorobiphenyl as sole sources of carbon by natural species of Ralstonia and Pseudomonas

Ralstonia sp. SA-3, Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 are natural strains with a novel capacity to utilize meta-substituted dichlorobiphenyls (diCBs) hitherto not known to serve as a sole source of carbon and energy for polychlorobiphenyl-degraders. In growth experiments, axenic cultures of isolates grew logarithmically on 3,3'-diCB with generation times that ranged insignificantly (t-test, P>0.05) from 30.4 to 33.8 h. Both 3-chlorobenzoate (3-CBA) and chloride produced as metabolites were recovered in non-stoichiometric quantities. The release of chloride by the cultures lagged substantially, indicating that the initial dioxygenase attack preceded cleavage of carbon-chloride bonds and that chloride must have been released from the chlorinated hydroxypentadienoate. In the case of 3,5-diCB, SA-3 and SA-6 metabolised this substrate primarily to 3,5-CBA. The lack of chloride in the culture media coupled with stoichiometric recovery of 3,5-CBA suggests that growth by these strains occurred predominantly at the expense of the unsubstituted phenyl ring. The unique metabolic properties of these three aerobic isolates point to their potential usefulness as seeds for bioremediation of PCBs polluted environments without the need for repeated inoculation or supplementation by a primary growth substrate such as biphenyl.     

Removal of 2,4-dichlorophenol by suspended and immobilized Bacillus insolitus

In this experiment, Bacillus insolitus was isolated and selected from a mixed culture that have been acclimated to chlorophenols. Decomposition of chlorophenolic compounds will be studied using this pure culture in both suspended and immobilized form. The results are: at lower initial concentrations of 2,4-dichlorophenol (10-50 mg/l), immobilized Bacillus insolitus shows a higher removal of 2,4-dichlorophenol than Bacillus insolitus in suspended growth. When the 2,4-dichlorophenol concentration becomes higher (50-200 mg/l), both immobilized and suspended Bacillus insolitus have approximately the same efficiency for removal of 2,4-dichlorophenol. Higher concentrations of 2,4-dichlorophenol are inhibitive to the growth of either suspended or immobilized Bacillus insolitus. At lower concentrations of 2,4-dichlorophenol, immobilized mixed culture may have the same removal efficiency of 2,4-dichlorophenol as immobilized pure culture of Bacillus insolitus. But with regard to the overall 2,4-dichlorophenol removal efficiency, immobilized pure culture is considered to be superior to immobilized mixed culture.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N2-ethyl-N?-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79-82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53-83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Short‐chain fatty acids affect cell‐association and invasion of HEp‐2 cells by Salmonella typhimurium

Abstract

This study demonstrates that the growth of S. typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell‐association and the ability to invade cultured HEp‐2 cells. Cell‐association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7. The results suggest that the growth rate of the culture may have affected cell‐association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell‐association and invasion. However, the results also suggest that the individual SCFA may play a role in modulating cell‐association and the invasion phenotype and the regulation of cell‐association and invasion by the SCFA was dependent on the concentration and the pH of the medium. Although the growth rates were similar for S. typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell‐association and invasion were observed among these cultures. Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79–82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53–83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Enrichment and isolation of endosulfan degrading and detoxifying bacteria

In the present study, degradation of endosulfan by a mixed culture isolated from a pesticide-contaminated soil was studied in batch experiments. After two weeks of incubation, the mixed culture was able to degrade 73% and 81% of alpha and beta endosulfan respectively. Endodiol was identified by GC/MS as degradation intermediate. The toxicity studies of endosulfan before and after degradation were carried out using micronucleus assay on human polymorphonuclear cells. The findings suggested that the metabolism of endosulfan isomers by the mixed culture was accompanied by significant reduction in the toxicity. Studies were also carried out to quantify the degradation potential of the individual species in the mixed bacterial culture. Two cultures identified by 16S rRNA as Stenotrophomonas maltophilia and Rhodococcus erythropolis were found to be responsible for majority of the degradation by the mixed culture. S. maltophilia showed better degradation efficiency compared to that by R. erythropolis. This is the first report of endosulfan degradation using the above-mentioned organisms.     

Biodegradation of mixed pesticides by mixed pesticide enriched cultures

This paper discusses the degradation kinetics of mixed (lindane, methyl parathion and carbofuran) pesticides by mixed pesticide enriched cultures (MEC) under various environmental conditions. The bacterial strains isolated from the mixed microbial consortium were identified as Pseudomonas aeruginosa (MTCC 9236), Bacillus sp. (MTCC 9235) and Chryseobacterium joostei (MTCC 9237). Batch studies were conducted to estimate the biokinetic parameters like the maximum specific growth rate (μ_max), Yield Coefficient (Y_T), half saturation concentration (K_s) and inhibition concentration (Ki) for individual and mixed pesticide enriched cultures. The cultures enriched in a particular pollutant always showed high growth rate and low inhibition in that particular pollutant compared to MEC. After seven weeks of incubation, mixed pesticide enriched cultures were able to degrade 72% lindane, 95% carbofuran and 100% of methyl parathion in facultative co-metabolic conditions. In aerobic systems, degradation efficiencies of lindane methyl parathion and carbofuran were increased by the addition of 2g L^{? 1} of dextrose. Though many metabolic compounds of mixed pesticides were observed at different time intervals, none of the metabolites were persistent. Based on the observed metabolites, a degradation pathway was postulated for different pesticides under various environmental conditions.