Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.     

The survival response of Escherichia coli K12 in a natural environment

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C.     

中高温污泥厌氧消化系统中微生物群落比较

【目的】结合中温与高温消化两者优势的两相厌氧消化工艺可能是推进污泥厌氧消化发展的重要方向,因此,探究和比较中温和高温污泥厌氧消化系统中微生物群落组成的异同具有重要意义。【方法】利用高通量测序技术检测中温和高温厌氧消化系统中细菌与古菌的16S r RNA基因序列信息和真菌的内转录间隔(ITS)序列信息,利用基因芯片(Geo Chip 5.0)检测病毒和病原菌致病基因的信息,以对比中温和高温条件下微生物群落在物种组成和功能基因层面上的异同。【结果】中温和高温条件下细菌和古菌在群落物种组成上存在显著差异,病毒和病原菌毒性基因也显著不同,而两种系统中真菌群落的物种组成相似且丰度相对较低。中温条件下产甲烷古菌和未分类微生物相对丰度较高,而高温条件下产酸及嗜热菌相对丰度较高,且高温消化后病毒和病原菌毒性基因相对丰度下降。微生物群落结构与COD、TS和VS有着显著相关性。【结论】微生物群落组成和功能基因在中高温的污泥厌氧消化系统中显著不同,从而解释了两个系统功能的差异。微生物群落的形成与进水参数相关,说明微生物对进水条件敏感。     

Improvement of sludge digestate biodegradability by thermophilic bioaugmentation

The sludge digestate stabilized by mesophilic anaerobic digestion was further degraded through thermophilic anaerobic digestion using 0–10 % (v/v) of thermophilic, proteolytic Coprothermobacter proteolyticus, and/or methanogenic granular sludge. The results demonstrated that the temperature shift to thermophilic condition promoted abiotic solubilization of proteins and reactivated the fermentative bacteria and methanogens indigenous in the sludge digestate, resulting in a final methane yield of 6.25 mmol-CH₄/g-volatile suspended solid (VSS) digestate. The addition of C. proteolyticus accelerated the hydrolysis and fermentation of proteins and polysaccharides in the digestate during the early stage of thermophilic anaerobic digestion and stimulated methane production by syntrophic cooperation with methanogenic granular sludge. In the treatment with granular sludge and inoculated with 10 % (v/v) of C. proteolyticus, a final methane yield of 7 mmol-CH₄/g-VSS digestate was obtained, and 48.4 % proteins and 27.0 % polysaccharides were degraded. The dissolved proteins were contributed by abiotic factor, C. proteolyticus, and indigenous digestate bacteria, respectively, by around 16, 28, and 56 %.     

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO₂-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.     

Ammonia inhibition in anaerobic digestion: A review

Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during anaerobic digestion process if it is available at high concentrations. Therefore, ammonia is regarded as a potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates such as manure or the organic fraction of municipal solid waste (OFMSW). Ammonia is produced through biological degradation of nitrogenous matter. Ammonium ion (NH₄⁺) and free ammonia (NH₃) are the two principal forms of inorganic ammonia nitrogen. Both forms can directly and indirectly cause inhibition in an anaerobic digestion system. Particularly, free ammonia (FAN) is a powerful inhibitor in an anaerobic digester above threshold concentrations. Process inhibition is related to the particular characteristics of the substrate to be anaerobically digested, pH, process temperature (mesophilic or thermophilic), type of the seed sludge (inoculum), the reactor configuration and to the concentrations of ammonium and ammonia. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.     

Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water

An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.     

Induction of Viable but Nonculturable Escherichia coli O157:H7 by High Pressure CO2 and Its Characteristics

The viable but nonculturable (VBNC) state is a survival strategy adopted by many pathogens when exposed to harsh environmental stresses. In this study, we investigated for the first time that whether high pressure CO₂ (HPCD), one of the nonthermal pasteurization techniques, can induce Escherichia coli O157:H7 into the VBNC state. By measuring plate counts, viable cell counts and total cell counts, E. coli O157:H7 in 0.85% NaCl solution (pH 7.0) was able to enter the VBNC state by HPCD treatment at 5 MPa and four temperatures (25°C, 31°C, 34°C and 37°C). Meanwhile, with the improvement of treatment temperature, the time required for E. coli O157:H7 to enter VBNC state would shorten. Enzymatic activities in these VBNC cells were lower than those in the exponential-phase cells by using API ZYM kit, which were also reduced with increasing the treatment temperature, but the mechanical resistance of the VBNC cells to sonication was enhanced. These results further confirmed VBNC state was a self-protection mechanism for some bacteria, which minimized cellular energetic requirements and increased the cell resistance. When incubated in tryptic soy broth at 37°C, the VBNC cells induced by HPCD treatment at 25°C, 31°C and 34°C achieved resuscitation, but their resuscitation capabilities decreased with increasing the treatment temperature. Furthermore, electron microscopy revealed changes in the morphology and interior structure of the VBNC cells and the resuscitated cells. These results demonstrated that HPCD could induce E. coli O157:H7 into the VBNC state. Therefore, it is necessary to detect if there exist VBNC microorganisms in HPCD-treated products by molecular-based methods for food safety.     

The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants

In this study, the abilities of two anaerobic digestion processes used for sewage sludge stabilization were compared for their ability to reduce the quantities of three genes that encode resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved with integrons (intI1). A two-stage, thermophilic/mesophilic digestion process always resulted in significant decreases in the quantities of tet(X) and intI1, less frequently in decreases of tet(O), and no net decrease in tet(A). The thermophilic stage was primarily responsible for reducing the quantities of these genes, while the subsequent mesophilic stage sometimes caused a rebound in their quantities. In contrast, a conventional anaerobic digestion process rarely caused a significant decrease in the quantities of any of these genes, with significant increases occurring more frequently. Our results demonstrate that anaerobic thermophilic treatment was more efficient in reducing quantities of genes associated with the spread of antibiotic resistance compared to mesophilic digestion.     

PCB and AOX removal in mesophilic and thermophilic sewage sludge digestion

In this study, a comparison of the biodegradation of adsorbed organic halogen compounds (AOX) and polychlorinated biphenyls (PCB) in thermophilic and mesophilic anaerobic digestion (seeded with waste activated sludge) at different hydraulic retention times (HRT 18, 22 and 26 days in the mesophilic digester and 8, 12, 18, 22 and 26 days in the thermophilic digester) was performed. Results obtained in this work showed an enhancement of both PCB and AOX biodegradation under thermophilic conditions. The total PCB removal efficiency was in the range of 59.4–83.5% under thermophilic conditions and 33.0–58.0% under mesophilic conditions. HRT played an important role in the digester performance since high working HRTs implied more reduction of the total PCB amount in the sludge. The total PCB content in the treated sludge under thermophilic conditions lied below the cut-off limit proposed in the 3rd draft of Directive presented to the European Commission [CEC, Working Document on Sludge (3rd Draft), Commission of the European Communities Directorate-General Environment, ENV.E.3/LM, Brussels, 27 April 2000]. Besides, a bioaccumulation of lightly chlorinated PCBs was detected in the mesophilic digester, which is in concordance with the theory that the PCBs are anaerobically biodegraded by means of a reductive dechlorination mechanism. On the other hand, the AOX removal efficiency was in the range of 40.4–50.3% for thermophilic conditions and 30.2–43.2% for mesophilic conditions. The AOX content in the treated sludge of both thermophilic and mesophilic digesters did not exceed the cut-off limit proposed in the 3rd draft [CEC, Working Document on Sludge (3rd Draft), Commission of the European Communities Directorate-General Environment, ENV.E.3/LM, Brussels, 27 April 2000]. Moreover, high HRTs promoted an improvement of the AOX removal capacity of the anaerobic digestion.     

Induction of the Viable but Nonculturable State of Ralstonia solanacearum by Low Temperature in the Soil Microcosm and Its Resuscitation by Catalase

Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.     

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.     

Thermophilic anaerobic digestion of sewage sludge: focus on the influence of the start-up. A review

The thermophilic anaerobic digestion (TAD) of sewage sludge has often been found to be less stable than mesophilic treatment. In comparison to mesophilic digesters, thermophilic reactors treating sludge are generally characterized by relatively high concentrations of volatile fatty acids (VFA) in the effluent along with poor effluent quality, indicating a lower level of process stability. However, reviewing the literature related to the procedure for obtaining a thermophilic inoculum, it seems that most of the problems associated with the instability and the accumulation of organic intermediates are the result of the manner in which the thermophilic sludge has been obtained. In this paper, the different options available for obtaining an anaerobic digester operating at thermophilic temperature (55°C) have been reviewed. In this light, rapid heating to the target temperature followed by the development of thermophilic microorganisms, which can be determined by VFA dropping to ≤500?mg acetic acid L^?1 before increasing the organic loading rate (OLR), has been determined the most suitable means of establishing TAD.     

Concentrations of Copper Thought To Be Toxic to Escherichia coli Can Induce the Viable but Nonculturable Condition

We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.     

Microbial populations and sludge characteristics in thermophilic aerobic sewage sludge digestion

Summary Estimates of bacterial numbers from raw sewage sludge and sludge treated by thermophilic aerobic digestion were compared with simple indicators of sludge quality and concentrations of potential substrates. Significant differences were found between sludge types for all but one of the variables examined (frequency of dividing cells). During a stable period of digestor operation, the average number of viable obligate thermophiles present in digested sludge (1.63 × 10⁶ ml^–1) was approximately 10²-fold greater than in feed sludge (1.10 × 10⁴ ml^–1). Total numbers of bacteria were slightly greater in digested sludge (3.24 × 10¹⁰ ml^–1) than in feed sludge (2.39 × 10 ml^–10), as were viable counts of bacteria at incubation temperatures of 37°C and 55°C. Significant correlation was found between viable counts of bacteria at 37°C and 55°C for digested sludge, and 65°C and 55°C for feed sludge. The numbers of obligate thermophiles present and the total of bacteria present were related to the temperature and pH of the digested sludge and inversely related to the numbers ofEscherichia coli and coliforms present, which were not detected at temperatures greater than 50°C.     

From mesophilic to thermophilic conditions: one-step temperature increase improves the methane production of a granular sludge treating agroindustrial effluents

Objectives

To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor.

Results

One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8–2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2–2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions.

Conclusions

More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

     

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 °C) and thermophilic (55 °C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 °C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 L_CH4/kg_VS (VS: volatile solids) and 121 L _CH4/kg_VS at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kg_{dry_biochar} was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.     

The use of ultrasound and gamma-irradiation as pre-treatments for the anaerobic digestion of waste activated sludge at mesophilic and thermophilic temperatures

The effect of ultrasound and gamma-irradiation used as pre-treatments for the anaerobic digestion of waste activated sludge at both mesophilic and thermophilic temperatures was examined. Untreated activated sludge was also subjected to anaerobic digestion at these temperatures as a control. The sonication time was 90 s using a Soniprep 150 (MSE Scientific Instruments) which operated at 23 kHz and had been adjusted to give an output of 47 W and the gamma-irradiation dose was 500 krad. The digesters were operated in a semi-continuous mode, being fed with fresh sludge every 24 h at hydraulic retention times (HRT) of 8, 10 and 12 days. Over the 24 h period the differences between the digesters, in terms of volatile solids (VS) reductions and biogas production, were not statistically significant for any particular set of conditions. Thermophilic digestion performed better than mesophilic digestion in terms of biogas production, VS reductions (except at HRT of 8 days) and specific methane yields and the optimum retention time was 10 days, at both temperatures. When gas production over the initial eight hours (probably the hydrolytic stage) was examined, it was found that the gas production rates for pre-treated sludges were higher than those for untreated sludges. This was most pronounced at thermophilic temperatures and a HRT of 10 days. Sonication did not affect the numbers of faecal coliforms in the sludge. However, gamma-radiation caused a 3-log reduction and, when coupled with mesophilic digestion, gave a product which contained < 100 g(-1) TS. Thermophilic anaerobic digestion produced sludges which contained < 1 g(-1) TS irrespective of any pre-treatment.