Aerobic Biodegradation of Mononitrotoluenes in Batch and Continuous Reactor Systems

Degradations of nitrotoluenes, individually and in a mixture, were carried out in batch and continuous aerobic reactors by a defined mixed microbial culture. The degradation rates and efficiencies of the isomers were evaluated in batch and continuous reactors. The results demonstrated that all the three-mononitrotoluene isomers were degraded simultaneously and completely in presence of excess oxygen. The nitro group position on the benzene ring influenced the degradation rates of the individual MNT isomers in batch systems. In the continuous biofilm reactor with a sufficient biocatalyst quantity, quality and excessive oxygen supply rate, the degradation rates of the mononitrotoluenes were almost identical as long as the compounds were present individually and their loading did not exceed the capacity of the catabolic master reaction. The microbial composition of the biofilm changed qualitatively and quantitatively during long-term continuous operation under aerobic non-aseptic conditions. This complex investigation resulted in data that can be applied for the scale-up procedure for field experiments.     

Fate of Chlortetracycline- and Tylosin-Resistant Bacteria in an Aerobic Thermophilic Sequencing Batch Reactor Treating Swine Waste

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25°C, 37°C, and 60°C decreased after treatment, but they were still abundant (10² to 10⁸ most probable number ml⁻¹) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25°C, 37°C, and 60°C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60°C and for tylosin-resistant populations grown at 37°C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.     

Impact of an Aerobic Thermophilic Sequencing Batch Reactor on Antibiotic-Resistant Anaerobic Bacteria in Swine Waste

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25°C and 37°C, resistant populations remained significant (10⁴ to 10⁵ most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37°C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.     

Population Changes in Enteric Bacteria and Other Microorganisms During Aerobic Thermophilic Windrow Composting

Composting of wastes from swine feeding operations was studied. The effects of the frequency of turning the wastes and addition of straw to improve the physical structure were studied to determine the most effective technique to rapidly increase the temperature and, consequently, destroy coliforms and Salmonella. Four different treatments were studied; the results showed that, with addition of 5% (wt/wt) straw and mechanical turning of the compost 20 times per week, the temperature reached 60 C within 3 days and enteric bacteria were destroyed within 14 days.     

Survival of Aerobic and Anaerobic Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total and anaerobic counts were ascertained on boneless, cooked, cubed, frozen chicken meat. We determined survival of aerobes and anaerobes in the natural flora after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and at 50, 85, and 100 C for 10 min. Total and anaerobic counts of bacteria in the rehydrated meat were established during storage of samples at 4, 22, and 37 C-until a spoilage odor was detected. Samples were also inoculated with Clostridium sporogenes and were dried and rehydrated at 100 C and stored at 37 C. Approximately 21% of the aerobes and 37% of the anaerobes survived drying and rehydration at room temperature. Many genera of aerobes, anaerobes, and facultative anaerobes survived drying and rehydration at 50 C; only sporeformers survived rehydration at 85 or 100 C. Low-temperature (4 C) storage of rehydrated meat produced ample shelf life (over 20 days), whereas storage at the higher temperature resulted in a shelf life of less than 30 hr. Approximately 81% of the C. sporogenes cells survived rehydration at 100 C and grew to over 10(7) cells within 40 hr. Our study presents additional data for adequate microbiological control in processing of freeze-dehydrated meat. Also, it points out the natural selection for sporeformers at high temperature of rehydration, stressing the need for consumer education in product handling for safety purposes.     

Effect of Pollution Load and Oxygen Availability on Thermophilic Aerobic Continuous Biodegradation of Potato Processing Wastewater

The possibility of enhancing the effectiveness of the biodegradation process of potato wastewater was evaluated. The effect of wastewater pollution load, dilution rate and oxygen supplementation during the thermophilic aerobic continuous utilization process on biodegradation of the main organic pollutants was investigated. Aeration performed using a stirred tank reactor (1.5 vvm, 300 rpm, 55 °C) proved to be not sufficient to provide aerobic conditions. During the continuous process, the amount of oxygen introduced into the wastewater was totally utilized by the microflora. The accumulation and decomposition of the fermentative metabolism products of the Bacillus mixed culture during oxygen limitation and supplementation were analyzed. Under microaerobic conditions (DOT < 1 %), a biosynthesis of acetic acid was observed up to a concentration of 6.5 g/dm³. After chemostat conditions were achieved, the level of acetic acid stabilized at 2.2 g/dm³. The addition of oxygen for aeration allowed a total decomposition of organic acids like acetic and lactic acid (the by‐products were synthesized from partially oxidized substrates) as well as an increase in COD reduction of the wastewater (from 66 % to 79 %). The periodic fortification of aeration with pure oxygen is proposed for critical situations during the continuous wastewater biodegradation when an unexpected inflow of highly polluted wastewater occurs in the system.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

Survival and Transmission of Salmonella enterica Serovar Typhimurium in an Outdoor Organic Pig Farming Environment

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g⁻¹) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n = 294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n = 10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.     

Plasmid-mediated serum resistance and alterations in the composition of lipopolysaccharides in Salmonella dublin

Survival rates of Salmonella dublin in rabbit serum after culture for 1 h at 37 degrees C were compared between a wild-type strain (5240) carrying a 50 MDa plasmid, a plasmid-cured strain (C524), and a cured strain containing the 50 MDa plasmid tagged with Tn1 (5241). Strain C524 was more susceptible to the bactericidal activity of normal serum than its parent strain 5240 (percentage survival less than 1% and 52.5 +/- 9.2%, respectively). On the other hand, the percentage survival of strain 5241 was significantly increased (90.4 +/- 4.0%), indicating that the reintroduction of the plasmid into the cured strain restored the serum resistance. Moreover, this change in the serum resistance properties correlated with changes in the neutral sugar composition of the lipopolysaccharides (LPS) of these strains, suggesting that the 50 MDa plasmid is necessary for O-side chain expression in the LPS of S. dublin.     

Prevalence and Identity of Cryptosporidium spp. in Pig Slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

A Novel Acidimicrobium Species in Continuous Cultures of Moderately Thermophilic, Mineral-Sulfide-Oxidizing Acidophiles

A novel species of Acidimicrobium appeared to be the predominant ferrous iron oxidizer in a mixed culture that effected the continuous, efficient extraction of nickel from a mineral concentrate at 49°C, but it was not isolated in pure culture. It outcompeted Acidimicrobium ferrooxidans, which was expected to have a major role in iron oxidation in reactors gassed with air, and was outnumbered at 49°C only by the sulfur-oxidizing Acidithiobacillus caldus. Sulfobacillus species were expected to compete with Acidimicrobium species when culture aeration was enriched with carbon dioxide, but they were a minor component of the populations with and without this enrichment. Sulfobacillus thermosulfidooxidans replaced the Acidimicrobium species and Acidithiobacillus caldus when the temperature was increased to 55°C.     

Survival of Salmonellas and Ascaris Suum Eggs in a Thermophilic Biogas Plant

In a continuous biogas plant, receiving manure from 200 dairy cows and 400 calves and young stock, survival of salmonellas and Ascaris suum eggs was studied. The bacteria and parasite eggs were kept in filter sacs in the manure that had a temperature of 55°C. No viable salmonellas or Ascaris suum eggs could be found after 24h in the digester. Survival of salmonellas and Ascaris suum eggs was also studied in the manure pit where the manure was stored after digestion. The temperature in the manure pit varied between 22–27°C. Salmonellas survived 35 but not 42 days. On day 56, when the experiments had to be stopped, 60% of the Ascaris eggs were viable.     

Effects of Physical Properties of Feed on Microbial Ecology and Survival of Salmonella enterica Serovar Typhimurium in the Pig Gastrointestinal Tract

A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of Salmonella enterica serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (P < 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of S. enterica serovar Typhimurium DT12 in content from the stomach (P < 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (P < 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of S. enterica serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (P < 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (P < 0.05) and colon (P < 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of Salmonella during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

Assessment of Bacterial Community Assembly Patterns and Processes in Pig Manure Slurry

The bacterial community assembly patterns and processes are poorly understood in pig manure slurry. We collected pig manure slurry samples during the winter and summer seasons from eight commercial pig farms in South Korea. The V3 region of 16S rRNA genes was PCR amplified and sequenced using paired-end Illumina technology for in-depth characterization of bacterial community. Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, and Tenericutes were the predominant bacterial phyla present in slurry samples. Bacterial taxonomic community composition was not influenced by the season; however, phylogenetic community composition was affected by seasonal variations. The community composition and diversity patterns were strongly influenced by pH. The bacterial diversity indices showed a unimodal relationship with pH. Phylogenetic signals were detected over only short phylogenetic distances, revealing that closely related bacterial operational taxonomic units (OTUs) tend to co-occur in the same environment; hence, they are ecologically similar. Across all samples, a niche-based process, through strong environmental filtering imposed by pH, primarily governed bacterial community assembly; however, in samples close to the neutral pH range, the role of environmental filtering was decreased due to neutral community assembly. In summary, pH emerged as the major physico-chemical variable in pig manure slurry that regulates the relative importance of niche-based and neutral processes in shaping the community assembly of bacteria.     

Survival of Salmonella senftenberg and Salmonella typhimurium in glassy and rubbery states of gelatin

Salmonella senftenberg and Salmonella typhimurium survived the production of gelatin sheets containing nutrient broth although there was some evidence of cell damage. Both strains survived but did not grow in glassy states with an a _w of 0.45–0.28 and rubbery states with an a _w of 0.93–0.96 for at least 28 d. Survival was less in intermediate states with an a _w between 0.55 and 0.74. The results suggest that salmonellas should be excluded from glassy state products in order to prevent salmonellosis.     

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis

Salmonella -induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin . We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.     

Growth, Survival and Characterization of cspA in Salmonella enteritidis Following Cold Shock

Salmonella enteritidis is a major foodborne microbial pathogen that can grow and survive at low temperatures for a considerable period of time. Increased survival was evidenced from a frozen S. enteritidis culture when treated at 10°C prior to freezing. Western blot analysis with Escherichia coli CspA antibody and analysis of radiolabeled proteins from S. enteritidis cultures after cold shock at 10°C and 5°C showed increased expression of a 7.4-kDa major cold shock protein, CS7.4, similar in size to that reported for E. coli. Cloning followed by nucleotide sequence analysis of the cspA gene from S. enteritidis showed a 100% nucleotide sequence identity in the promoter elements (−35 and −10) and the amino acid sequence encoded by the open reading frame (ORF) with the E. coli cspA gene. However, the differences in the nucleotide sequences between E. coli and S. enteritidis cspA genes in the putative repressor protein binding domain, the fragment 7, and in various segments throughout the upstream 0.642-kbp DNA may contribute to the expression of CS7.4 at less stringent temperatures in S. enteritidis. As in E. coli, the actual role of CS7.4 in protecting S. enteritidis from the damaging effects of cold or freezing temperatures is not yet understood. Received: 14 March 1997 / Accepted: 10 July 1997