Growth of Dunaliella tertiolecta and associated bacteria in photobioreactors

The aim of this study was to test three flat-plate photobioreactor configurations for cultivation of marine green alga Dunaliella tertiolecta under non-axenic growth conditions and to characterize and quantify the associated bacteria. The photobioreactor cultivations were conducted using tap water-based media. Static mixers intended to enhance mixing and light utilization did not generally increase algal growth at the low light intensities used. The maximum biomass concentration (measured as volatile suspended solids) and maximum specific growth rate achieved in the flat plate with no mixer were 2.9?g?l(-1) and 1.3?day(-1), respectively. Based on quantitative polymerase chain reaction, bacterial growth followed the growth of D. tertiolecta. Based on 16S rDNA amplification and denaturing gradient gel electrophoresis profiling, heterotrophic bacteria in the D. tertiolecta cultures mainly originated from the non-axenic algal inocula, and tap water heterotrophs were not enriched in high chloride media (3?% salinity). Bacterial communities were relatively stable and reproducible in all flat-plate cultivations and were dominated by Gammaproteobacteria, Flavobacteria, and Alphaproteobacteria.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

Diversity and functional traits of culturable microbiome members,including cyanobacteria in the rice phyllosphere

The diversity and abundance of culturable microbiome members of the rice phyllosphere was investigated using cv. Pusa Punjab Basmati 1509. Both diversity and species richness of bacteria were significantly higher in plants in pots in a semi‐controlled environment than those in fields. Application of fertilisers reduced both diversity and species richness in field‐grown plants under a conventional flooded system of rice intensification (SRI) and in dry‐seeded rice (DSR) modes. Sequence analyses of 16S rDNA of culturable bacteria, those selected after amplified ribosomal DNA restriction analysis (ARDRA), showed the dominance of α‐proteobacteria (35%) and actinobacteria (38%); Pantoea, Exiguobacterium and Bacillus were common among the culturable phyllospheric bacteria. About 34% of 83 culturable bacterial isolates had higher potential (>2 μg·ml^?1) for indole acetic acid production in the absence of tryptophan. Interestingly, the phyllosphere bacterial isolates from the pot experiment had significantly higher potential for nitrogen fixation than isolates from the field experiment. Enrichment for cyanobacteria showed both unicellular forms and non‐heterocystous filaments under aerobic as well as anaerobic conditions. PCR‐DGGE analysis of these showed that aerobic and anaerobic conditions as well as the three modes of cultivation of rice in the field strongly influenced the number and abundance of phylotypes. The adaptability and functional traits of these culturable microbiome members suggest enormous diversity in the phyllosphere, including potential for plant growth promotion, which was also significantly influenced by the different methods of growing rice.     

In situ ecological development of a bacteriogenic iron oxide-producing microbial community from a subsurface granitic rock environment

The initial development and diversity of an in situ subsurface microbial community producing bacteriogenic iron oxides (BIOS) were investigated at the initiation of biofilm growth (2‐month period) and after a 1‐year period of undisturbed growth. Water chemistry data, samples of iron encrusted biofilm material and groundwater were collected from BRIC (BIOS reactor, in situ, continuous flow) apparatuses installed 297 m below sea level at the Äspö Hard Rock Laboratory (HRL) in south eastern Sweden. Comparisons between the BIOS BRIC system and an anaerobic control (AC) BRIC revealed that water mixing at the inflow leads to profuse development of BIOS related to a slightly elevated level of O₂ (up to 0.3 mg L^?1 at the transition zone between BIOS development and non‐development) and elevated Eh (>120 mV) in the first 70 mm of water depth. Decreases in dissolved and particulate iron were connected to the visible appearance of BIOS biofilms. The basic phylogenetic diversity of this site was evaluated using amplified ribosomal DNA restriction enzyme analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE) and partial sequencing of 16S rDNA. From 67 clones that were positive for 16S rDNA inserts, a total of 42 different ARDRA profiles were recognized, representing four bacterial phyla and 14 different metabolic lifestyles. DGGE profiles indicated that there are differences in the representative bacteria when considering either BIOS biofilms or groundwater. DGGE also indicated that the DNA extraction protocols and any polymerase chain reaction biases were consistent. Bacterial metabolic groups associated with indirect metal adsorption and reduction along with bacteria utilizing many alternative electron acceptors were strongly represented within the clones. This study indicates that the microbial diversity of BIOS is greater than previously thought.     

THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE) REQUIRES MARINE BACTERIA FOR GROWTH1

Interactions with the bacterial community are increasingly considered to have a significant influence on marine phytoplankton populations. Here we used a simplified dinoflagellate‐bacterium experimental culture model to conclusively demonstrate that the toxic dinoflagellate Gymnodinium catenatum H. W. Graham requires growth‐stimulatory marine bacteria for postgermination survival and growth, from the point of resting cyst germination through to vegetative growth at bloom concentrations (10³ cells · mL^?1). Cysts of G. catenatum were germinated and grown in unibacterial coculture with antibiotic‐resistant or antibiotic‐sensitive Marinobacter sp. DG879 or Brachybacterium sp., and with mixtures of these two bacteria. Addition of antibiotics to cultures grown with antibiotic‐sensitive strains of bacteria resulted in death of the dinoflagellate culture, whereas cultures grown with antibiotic‐resistant bacteria survived antibiotic addition and continued to grow beyond the 21 d experiment. Removal of either bacterial type from mixed‐bacterial dinoflagellate cultures (using an antibiotic) resulted in cessation of dinoflagellate growth until bacterial concentration recovered to preaddition concentrations, suggesting that the bacterial growth factors are used for dinoflagellate growth or are labile. Examination of published reports of axenic dinoflagellate culture indicate that a requirement for bacteria is not universal among dinoflagellates, but rather that species may vary in their relative reliance on, and relationship with, the bacterial community. The experimental model approach described here solves a number of inherent and logical problems plaguing studies of algal‐bacterium interactions and provides a flexible and tractable tool that can be extended to examine bacterial interactions with other phytoplankton species.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

Carbon dioxide biofixation by Chlorella vulgaris at different CO2 concentrations and light intensities

In order to develop an effective CO₂ mitigation process using microalgae for potential industrial application, the growth and physiological activity of Chlorella vulgaris in photobioreactor cultures were studied. C. vulgaris was grown at two CO₂ concentrations (2 and 13% of CO₂ v/v) and at three incident light intensities (50, 120 and 180 μmol m^?2 s^?1) for 9 days. The measured specific growth rate was similar under all conditions tested but an increase in light intensity and CO₂ concentration affected the biomass and cell concentrations. Although carbon limitation was observed at 2% CO₂, similar cellular composition was measured in both conditions. Light limitation induced a net change in the growth behavior of C. vulgaris. Nitrogen limitation seemed to decrease the nitrogen quota of the cells and rise the intracellular carbon:nitrogen ratio. Exopolysaccharide production per cell appeared to be affected by light intensity. In order to avoid underestimation of the CO₂ biofixation rate of the microalgae, exopolysaccharide production was taken into account. The maximum CO₂ removal rate (0.98 g CO₂ L^?1 d^?1) and the highest biomass concentration (4.14 g DW L^?1) were determined at 13% (v/v) CO₂ and 180 μmol m^?2 s^?1. Our results show that C. vulgaris has a real potential for industrial CO₂ remediation.     

<Emphasis Type="Italic">Chlorella vulgaris</Emphasis> (SAG 211-12) biofilm formation capacity and proposal of a rotating flat plate photobioreactor for more sustainable biomass production

Difficulties and cost of suspended microalgal biomass harvest and processing can be overcome by cultivating microalgae as biofilms. In the present work, a new photoautotrophic biofilm photobioreactor, the rotating flat plate photobioreactor (RFPPB), was developed aiming at a cost-effective production of Chlorella vulgaris (SAG 211-12), a strain not frequently referred in the literature but promising for biofuel production. Protocols were developed for evaluating initial adhesion to different materials and testing the conditions for biofilm formation. Polyvinyl chloride substrate promoted higher adhesion and biofilm production, followed by polypropylene, polyethylene, and stainless steel. The new RFPPB was tested, aiming at optimizing incident light utilization, minimizing footprint area and simplifying biomass harvesting. Tests show that the photobioreactor is robust, promotes biofilm development, and has simple operation, small footprint, and easy biomass harvest. Biomass production (dry weight) under non-optimized conditions was 3.35 g m^?2, and areal productivity was 2.99 g m^?2 day^?1. Lipid content was 10.3% (dw), with high PUFA content. These results are promising and can be improved by optimizing some operational parameters, together with evaluation of long-term photobioreactor maximum productivity.     

Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non‐Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture‐dependent methods. Moreover, production temperature played a more decisive role on the formation of micro‐organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR–DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

Diet shapes the ability of human intestinal microbiota to degrade phytate – in vitro studies

Aims

Investigation of intestinal bacterial groups involved in phytate degradation and the impact of diets with different phytate contents on phytase activity.

Methods and Results

Faecal samples of adults on conventional (n = 8) or vegetarian (n = 8) diets and breastfed infants (n = 6) were used as an inoculum for modified media supplemented with phytate. Populations of Gram‐positive anaerobes (GPA), lactic acid bacteria (LAB), Proteobacteria–Bacteroides (P‐B), coliforms and anaerobes were studied. The PCR‐DGGE analysis revealed a random distribution of DGGE profiles in the dendrograms of GPA, P‐B and coliforms, and a partially diet‐specific distribution in the DGGE dendrograms of LAB and anaerobes. The degradation of phytic acid (PA) was determined with HPLC method in supernatants of the cultures. Regardless of the diet, the Gram‐positive anaerobes and LAB displayed the lowest ability to degrade phytate, whereas the coliforms and P‐B cultures produced higher amounts of intermediate myo‐inositol phosphates. Bacterial populations grown in a nonselective medium were the most effective ones in phytate degradation. It was the vegetarians' microbiota that particularly degraded up to 100% phytate to myo‐inositol phosphate products lower than InsP₃.

Conclusions

A diet rich in phytate increases the potential of intestinal microbiota to degrade phytate. The co‐operation of aerobic and anaerobic bacteria is essential for the complete phytate degradation.

Significance and Impact of the Study

This study provides insights on the effect of diet on specific metabolic activity of human intestinal microbiota.     

Use of quantitative real-time PCR to monitor population dynamics of ammonia-oxidizing bacteria in batch process

A quantitative real-time PCR (QPCR) assay with the TaqMan system was used to quantify 16S rRNA genes of β-proteobacterial ammonia-oxidizing bacteria (AOB) in a batch nitrification bioreactor. Five different sets of primers, together with a TaqMan probe, were used to quantify the 16S rRNA genes of β-proteobacterial AOB belonging to the Nitrosomonas europaea, Nitrosococcus mobilis, Nitrosomonas nitrosa, and Nitrosomonas cryotolerans clusters, and the genus Nitrosospira. We also used PCR followed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing of their 16S rRNA genes to identify the AOB species. Seed sludge from an industrial wastewater treatment process controlling high-strength nitrogen wastewater (500 mg/L NH₄ ⁺–N) was used as the inoculum for subsequent batch experiment. The Nitrosomonas nitrosa cluster was the predominant AOB (2.3 × 10⁵ copies/mL) in the start-up period of the batch experiment. However, from the exponential growth period, the Nitrosomonas europaea cluster was the most abundant AOB, and its 16S rRNA gene copy number increased to 8.9 × 10⁶ copies/mL. The competitive dominance between the two AOB clusters is consistent with observed differences in ammonia tolerance and substrate affinity. Analysis of the DGGE results indicated the presence of Nitrosomonas europaea ATCC19718 and Nitrosomonas nitrosa Nm90, consistent with the QPCR results.     

Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR

Aims: In this article, a quantitative real‐time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time‐consuming and not always accurate. Methods and Results: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non‐target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C_t values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10–14 CFU ml^?1 in either cola or beer and at levels of 9·4–25·0 CFU ml^?1 in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. Conclusions: The results indicate that real‐time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. Significance and Impact of the Study: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.     

Study of bioaerosols and site influence in the La Plata area (Argentina) using conventional and DNA (fingerprint) based methods

The present study approaches the characterization of seasonal samples of bioaerosols taken during 2005–2006 in rural, urban, industrial, coastal and residential sites within La Plata area, Buenos Aires, Argentina. Culturable airborne fungal and bacterial communities were collected on DG18 agar and R2 Agar plates respectively, using a single-stage SKC sampling device. Fungal genera were identified based on their micro- and macro-morphological characteristics. Bacterial populations were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR 16SrDNA bacterial amplification. Geometric mean concentration of bacteria and fungi ranged from 10 to 10³ CFU m⁻³ and were comparable with those from other reports. No differences were observed among sites for fungi and Cladosporium sp. was predominant, with 85.7% of total concentration counts; secondary genera that contributed were Alternaria (6.7%), Penicillium (1.8%), Aspergillus (1.3%), Epicoccum (1.0%) and the group of yeasts (1.9%). The dominance of Cladosporium in all sites suggests that the most abundant fungal aerosol was neither significantly affected nor primarily generated by any anthropogenic area source more than the natural ambience present in the La Plata area. With regard to bacteria, the results showed on several occasions differences in concentration among sites during sampling events, but these differences were not observed when the community structure was analyzed by means of DGGE. Bacterial DGGE banding profiles from all sites revealed the existence of a relatively diversified, culture-based airborne community. Construction of similarity dendrograms exposed a distribution of site samples in which replicates intra-site equalled those encountered among sites, rendering substantial inference of site distinction unfeasible.     

The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil

Aims: To study how repeated applications of an herbicide bromoxynil to a soil, mimicking the regime used in the field, affected the degradation of the compound and whether such affects were reflected by changes in the indigenous bacterial community present. Methods and Results: Bromoxynil degradation was monitored in soil microcosms using HPLC. Its impact on the bacterial community was determined using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of five bacterial taxa (Pseudomonads, Actinobacteria, α‐Proteobacteria, Acidobacteria and nitrifying bacteria). Three applications of 10 mg kg^?1 of bromoxynil at 28‐day intervals resulted in rapid degradation, the time for removal of 50% of the compound decreasing from 6·4 days on the first application to 4·9 days by the third. Bacterial population profiles showed significant similarity throughout the experiment. With the addition of 50 mg kg^?1 bromoxynil to soil, the degradation was preceded by a lag phase and the time for 50% of the compound to be degraded increased from 7 days to 28 days by the third application. The bacterial population showed significant differences 7 days after the final application of bromoxynil that correlated with an inhibition of degradation during the same period. Conclusions: These analyses highlighted that the addition of bromoxynil gave rise to significant shifts in the community diversity and its structure as measured by four abundant taxa, when compared with the control microcosm. These changes persisted even after bromoxynil had been degraded. Significance and Impact of the Study: Here we show that bromoxynil can exert an inhibitory effect on the bacterial population that results in decreased rates of degradation and increased persistence of the compound. In addition, we demonstrate that molecular approaches can identify statistically significant changes in microbial communities that occur in conjunction with changes in the rate of degradation of the compound in the soil.     

Low‐temperature (9°C) AMD treatment in a sulfidogenic bioreactor dominated by a mesophilic Desulfomicrobium species

The possibilities for the treatment of low‐temperature mine waste waters have not been widely studied. The amenability of low‐temperature sulfate reduction for mine waste water treatment at 9°C was studied in a bench‐scale fluidized‐bed bioreactor (FBR). Formate was used as the electron and carbon source. The first influent for the FBR was acidic, synthetic waste water containing iron, nutrients, and sulfate, followed by diluted barren bioleaching solution (DBBS). The average sulfate reduction rates were 8 mmol L^?1 day^?1 and 6 mmol L^?1 day^?1 with synthetic waste water and DBBS, respectively. The corresponding specific activities were 2.4 and 1.6 mmol SO g VSS^?1 day^?1, respectively. The composition of the microbial community and the active species of the FBR was analyzed by extracting the DNA and RNA, followed by PCR‐DGGE with the universal bacterial 16S rRNA gene primers and dsrB‐primers specific for sulfate‐reducing bacteria. The FBR microbial community was simple and stable and the dominant and active species belonged to the genus Desulfomicrobium. In summary, long‐term operation of a low‐temperature bioreactor resulted in enrichment of formate‐utilizing, psychrotolerant mesophilic sulfate reducing bacteria. Biotechnol. Bioeng. 2009; 104: 740–751 © 2009 Wiley Periodicals, Inc.     

Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions

Aim: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. Methods and Results: Nitrate‐reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene–based PCR‐DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. Conclusions: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene‐ and benzoate‐degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. Significance and Impact of the Study: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.     

Midgut bacterial communities in the giant Asian honeybee (Apis dorsata) across 4 developmental stages: A comparative study

Bacterial communities are known to play important roles during the developmental stages of insects, but current knowledge of bacteria associated with the midgut of Apis dorsata, the giant Asian honeybee, is limited. Using polymerase chain reaction‐denaturing gradient gel electrophoresis analysis (PCR‐DGGE) and 16S rRNA sequencing, the aim of this study was to determine the dynamics of bacterial community structure across four A. dorsata life stages in different geographical locations. The results reveal that bacterial diversity increased as the bee progressed through larval stage to newly emerged worker and old worker. However, in the pupal stage, no bands identified as bacteria could be observed. Overall, 2 bacterial phyla (Proteobacteria and Firmicutes) and 4 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacilli) were identified, but the frequency varied among the different stages and locations. The classes of Gammaproteobacteria and Bacilli dominated among larval, newly emerged worker and old worker developmental stages.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.     

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration

Aims: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. Methods and Results: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). Moreover, species‐specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species‐specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. Conclusions: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. Significance and Impact of the Study: This study provides important information that may be used to extend the shelf life of kava beverages.