Fingerprinting and Identification of Bacteria Present in UASB Granules Used to Treat Winery, Brewery, Distillery or Peach-lye Canning Wastewater

The effective operation of the anaerobic digestion process in an upflow anaerobic sludge blanket (UASB) bioreactoris dependent on the microbial composition of the UASB granules. The granules contain a consortium of bacteria,with a specific metabolic function for each group, contributing to the overall efficiency and stability of thebioreactor. The aim of this study was to fingerprint and identify the bacteria present in four different types of SouthAfrican UASB granules that are used to treat winery, brewery, distillery and peach-lye canning wastewaters. Thiswas done by combining conventional microbiological platings with PCR-based denaturing gradient gelelectrophoresis (DGGE) and DNA sequence analysis. Each granule type showed distinct PCR-based DGGEfingerprints with unique bands, while other bands were found to be present in all the granules, regardless of thewastewater being treated. Sixty-eight different bacteria (40 pure isolates and 28 clones) were partially sequencedand identified from the winery, brewery, distillery and peach-lye canning granules. Thirty-five percent of theidentified bacteria represented the unculturable bacteria and 65% represented the culturable bacteria, whichincluded members of the following genera: Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes,Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas,Nitrospira, Synergistes, Rhodococcus, Rhodocyclus and Syntrophobacter. A DGGE marker was successfullyconstructed, representing members of the bacterial consortium in UASB granules

PCR-based DGGE Identification of Bacteria Present in Pasteurised South African Fruit Juices

The contamination of pasteurised fruit juice products by thermophilic acidophilic bacteria (TAB) has become aconcern for producers. The aim of this study was to identify the bacteria present in South African fruit juices beforeand after pasteurisation, using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis(DGGE). Alicyclobacillus acidoterrestris was found to be present in apple, pear, white grape and aloe vera juice.White grape juice was found to contain Alicyclobacillus pomorum, while two uncultured bacteria in the orange,apple, mango and pear juices were presumptively identified as members of the genus Bacillus, and one unculturedbacteria was identified as being closely related to Alcaligenes faecalis. The results emphasise the need for rapid andaccurate detection of TAB in food products

PCR and DGGE Detection Limits for Wine Spoilage Microbes

In this study the culture-independent technique, polymerase chain reaction (PCR)-denaturing gradient gelelectrophoresis (DGGE), was investigated for the early detection and identification of possible spoilage microbesin wine. PCR and DGGE conditions were successfully optimised with the universal primers HDA1GC and HDA2,the bacteria-specific primers WBAC1GC-WBAC2, and the yeast-specific primers NL1GC and LS2. PCR and DGGEdetection limits were determined for Lactobacillus plantarum, Pediococcus pentosaceus, Acetobacter pasteurianusand Brettanomyces bruxellensis when inoculated into sterile saline solution (SSS) and white wine at 106 cfu/mLrespectively. PCR detection limits were more sensitive (101 to 102 cfu/mL) than DGGE detection limits (101 to 104 cfu/mL), with the exception of B. bruxellensis, which had higher PCR and DGGE detection limits than the other referencemicrobes. PCR-DGGE analysis was also used successfully to detect and identify Lb. plantarum, A. pasteurianus andB. bruxellensis at a concentration of 108 cfu/mL as part of mixed populations in SSS and white wine. PCR detectionlimits of 101 cfu/mL were determined for all three reference microbes in mixed populations. The DGGE detectionlimits were higher for mixed populations when compared to single strains

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml -1) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml-1, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment. © 2008 The Authors.Articl

Identification of bacterial species on Lippia multiflora herbal tea leaves and the influence of steam pasteurization

The consumption of herbal teas is an increasing phenomenon among tea consumers globally. Some of these herbal teas are not pre-treated to reduce their microbial load before consumption, and thus constitute a health risk to consumers. In this study, the effect of steam pasteurization, at >99 °C for 2.5 min, on the microbial load of Lippia multiflora herbal tea leaves was evaluated. Microbial enumeration was conducted on potato dextrose agar, plate count agar, violet red bile agar, yeast peptone dextrose agar, and DeMan-Rogosa-Sharpe agar. Morphologically distinct colonies were isolated, sub-cultured and their Gram reaction recorded. These bacteria were identified to the species level using 16S ribosomal DNA sequence data. Most of the bacteria identified belonged to the genus Bacillus. One species each from the genera Pantoea and Kocuria was also identified, but only the Bacillus species survived the steam pasteurization treatment. Coliform bacteria detected prior to pasteurization were not detected after the steam treatment. Steam pasteurization reduced the microbial load from 104 to 102 c.f.u.g-1and it is potentially an effective method to treat L. multiflora herbal teas prior to consumption. It is important to note that the steam treatment should complement good agricultural and hygienic practices rather than replace them, as some bacteria can survive this treatment. © 2010 Springer Science+Business Media B.V.The consumption of herbal teas is an increasing phenomenon among tea consumers globally. Some of these herbal teas are not pre-treated to reduce their microbial load before consumption, and thus constitute a health risk to consumers. In this study, the effect of steam pasteurization, at >99 °C for 2.5 min, on the microbial load of Lippia multiflora herbal tea leaves was evaluated. Microbial enumeration was conducted on potato dextrose agar, plate count agar, violet red bile agar, yeast peptone dextrose agar, and DeMan-Rogosa-Sharpe agar. Morphologically distinct colonies were isolated, sub-cultured and their Gram reaction recorded. These bacteria were identified to the species level using 16S ribosomal DNA sequence data. Most of the bacteria identified belonged to the genus Bacillus. One species each from the genera Pantoea and Kocuria was also identified, but only the Bacillus species survived the steam pasteurization treatment. Coliform bacteria detected prior to pasteurization were not detected after the steam treatment. Steam pasteurization reduced the microbial load from 104 to 102 c.f.u.g-1and it is potentially an effective method to treat L. multiflora herbal teas prior to consumption. It is important to note that the steam treatment should complement good agricultural and hygienic practices rather than replace them, as some bacteria can survive this treatment. © 2010 Springer Science+Business Media B.V.ArticleArticl

PCR-based DGGE identification of bacteria present in pasteurised South African fruit juices

The contamination of pasteurised fruit juice products by thermophilic acidophilic bacteria (TAB) has become a concern for producers. The aim of this study was to identify the bacteria present in South African fruit juices before and after pasteurisation, using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). Alicyclobacillus acidoterrestris was found to be present in apple, pear, white grape and aloe vera juice. White grape juice was found to contain Alicyclobacillus pomorum, while two uncultured bacteria in the orange, apple, mango and pear juices were presumptively identified as members of the genus Bacillus, and one uncultured bacteria was identified as being closely related to Alcaligenes faecalis. The results emphasise the need for rapid and accurate detection of TAB in food products.Articl

Occurrence of Alicyclobacillus in the fruit processing environment - A review

Concentrated fruit products have a significant place in modern consumption markets and are valuable semi-prepared food components to the bakery, dairy, confectionary, canning, baby food, frozen food, distilling and beverage industries. There is continuous pressure on the beverage industry to improve the quality of concentrated fruit products in order for reconstituted fruit beverages to compete with beverages that are made from fresh fruits. In recent years, Alicyclobacillus spp. have become a major concern to the beverage industry worldwide as many high-acid, concentrated fruit products have been found to be contaminated with these spoilage microbes. The thermo-acidophilic nature of alicyclobacilli and highly resistant endospores allows for their survival during the production of concentrated fruit products. Under favourable conditions, endospores can germinate and multiply to numbers high enough to cause spoilage and product deterioration through the production of chemical taint compounds. It is imperative to understand the nature of Alicyclobacillus within the fruit concentrate processing environment so as to develop effective control strategies and to prevent spoilage in juice and beverage products that are reconstituted from fruit concentrates. This paper reviews the occurrence of alicyclobacilli in the fruit processing environment, control measures, as well as detection, identification and standardised test methods that are currently used for Alicyclobacillus in concentrated fruit products. © 2011 Elsevier B.V.Revie

Fingerprinting and identification of bacteria present in UASB granules used to treat winery, brewery, distillery or peach-lye canning wastewater

The effective operation of the anaerobic digestion process in an upflow anaerobic sludge blanket (UASB) bioreactor is dependent on the microbial composition of the UASB granules. The granules contain a consortium of bacteria, with a specific metabolic function for each group, contributing to the overall efficiency and stability of the bioreactor. The aim of this study was to fingerprint and identify the bacteria present in four different types of South African UASB granules that are used to treat winery, brewery, distillery and peach-lye canning wastewaters. This was done by combining conventional microbiological platings with PCR-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules, regardless of the wastewater being treated. Sixty-eight different bacteria (40 pure isolates and 28 clones) were partially sequenced and identified from the winery, brewery, distillery and peach-lye canning granules. Thirty-five percent of the identified bacteria represented the unculturable bacteria and 65% represented the culturable bacteria, which included members of the following genera: Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus and Syntrophobacter. A DGGE marker was successfully constructed, representing members of the bacterial consortium in UASB granules.Articl

PCR-based denaturing gradient gel electrophoretic evaluation of changes in the non-methanogenic population of stressed upflow anaerobic sludge blanket granules

The performance of upflow anaerobic sludge blanket (UASB) bioreactors is influenced by the composition of the substrate and the microbial species present in the granules. The aim of this study was to determine if a change in the structure of the non-methanogenic microbial community takes place when UASB brewery granules are subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium. The granules that were stressed with glucose medium did not show changes in the microbial consortium regardless of the increase in the glucose concentrations. The polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) method was successfully applied to show changes in the structure of the microbes present in UASB granules that were cultivated under different environmental conditions. © 2006 Springer Science+Business Media B.V.Articl