Ramesh Goel

Factors affecting bulk to total bacteria ratio in drinking water distribution systems

Bacteria in drinking water systems can grow in bulk water and as biofilms attached to pipe walls, both causing regrowth problems in the distribution system. While studies have focused on evaluating the factors influencing the bacteria in bulk water and in biofilms separately, there is a need for understanding biofilm characteristics relative to the bulk water phase. The current study evaluated the effects of chlorine and residence time on the presence of culturable bacteria in biofilms relative to that in bulk water. The results showed that when no chlorine residual was present in the system, the median ratio of bulk to total bacteria was 0.81, indicating that 81% of the bacteria were present in bulk water, whereas only 19% were present in the biofilm. As chlorine concentration increased to 0.2, 0.5, and 0.7 mg/L, the median percentage of bacteria present in bulk water decreased to 37, 28, and 31, respectively. On the other hand, as the residence times increased to 8.2, 12, 24, and 48h, the median percentage of bacteria present in bulk water increased to 7, 37, 58, and 88, respectively, in the presence of a 0.2mg/L chlorine residual. The common notion that biofilms dominate the distribution system is not true under all conditions. These findings suggest that bulk water bacteria may dominate in portions of a distribution system that have a low chlorine residual.

Kinetic study of electrolytic ammonia removal using Ti/IrO2 as anode under different experimental conditions.

This study reports electrolytic degradation and kinetic modeling of electrolytic degradation of ammonia using Ti/IrO(2). The experimental and modeling results are compared with those previously obtained using Ti/RuO(2) anode. Synthetic solution containing predetermined ammonia concentration and the raw municipal wastewater collected after the aerobic or the anaerobic treatment were used in different sets of experiments. The experimental conditions varied for electrolytic degradation of ammonia present in the synthetic feed were initial chloride concentration, initial pH and applied current density. The results show that the ammonia removal followed pseudo zero-order kinetics. The current density (j) and the initial Cl(-) concentration ([Cl(-)]) affected the constant of the pseudo zero-order kinetics, expressed as k(Ti/IrO(2))=0.0020[Cl-]j + 0.4848 and k(Ti/RuO(2))=0.0026[Cl-]j - 0.7417. The ammonia removal rate averaged at 8.5 using Ti/IrO(2) and was comparable to the rate (11.7 mg N L(-1)h(-1)) obtained using Ti/RuO(2) anode, at 15.4 mA cm(-2) current density, 300 mg L(-1)Cl(-) and pH of 7. Higher current density and as well as the chloride concentration resulted in greater ammonia degradation. On the other hand, pH did not seem to have any effect on electrolytic degradation of ammonia. More than 95% ammonia degradation in municipal wastewater effluents collected after aerobic or anaerobic treatment was achieved. The ammonia nitrogen in the electrolytically treated wastewater effluents collected at the end of aerobic and anaerobic environments were 0.9, 0.5 mg N L(-1) using Ti/IrO(2) and 0.5, 0.2 mg N L(-1) by using Ti/RuO(2) anodes, respectively.

Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly isolated lytic bacteriophage

In a previous paper, the first ever application of lytic bacteriophage (virus)-mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium was demonstrated. In this work we extended the biocontrol application to another predominant filamentous bacterium, Sphaerotilus natans, notoriously known to cause filamentous bulking in wastewater treatment systems. Very similar to previous study, one lytic bacteriophage was isolated from wastewater that could infect S. natans and cause lysis. Significant reduction in sludge volume index and turbidity of the supernatant was observed in batches containing S. natans biomass following addition of lytic phages. Microscopic examination confirmed that the isolated lytic phage can trigger the bacteriolysis of S. natans. This extended finding further strengthens our hypothesis of bacteriophage-based biocontrol of overgrowth of filamentous bacteria and the possibility of phage application in activated sludge processes, the worlds widely used wastewater treatment processes.This research demonstrates the first ever application of lytic bacteriophage (virus) mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium. Bacteriophages are viruses that specifically infect bacteria only. The lytic phage specifically infecting H. hydrossis was isolated from the mixed liquor of a local wastewater treatment plant. The isolated bacteriophage belongs to the Myoviridae family with a contractile tail (length-126 nm; diameter-18 nm) and icosahedral head (diameter-81 nm). Titer of the isolated phage with H. hydrossis was calculated to be 5.2 ± 0.3 × 10(5) PFU/mL and burst size was found to be 105 ± 7 PFU/infected cell. The phage was considerably stable after exposure to high temperature (42 °C) and pH between 5 and 8, emphasizing that it can withstand the seasonal/operational fluctuations under real-time applications. Phage to host (bacteria) ratio for the optimal infection was found to be 1:1000 with ∼54% host death. The isolated phage showed no cross infectivity with other bacteria most commonly found in activated sludge systems, thus validating its suitability for biocontrol of filamentous bulking caused by H. hydrossis. Following the phage application, successful reduction in sludge volume index (SVI) from 155 to 105 was achieved, indicating improved biomass settling. The application of phage did not affect nutrient removal efficiency of the biomass, suggesting no collateral damage. Similar to phage therapy in medical applications, phage-mediated biocontrol holds a great potentiality for large-scale applications as economic agent in the mitigation of several water, wastewater and environmental problems. Present study in this direction is a novel effort.

Evaluation of simultaneous nutrient removal and sludge reduction using laboratory scale sequencing batch reactors

The treatment and disposal of excess sludge has been a rising challenge for wastewater treatment plants worldwide. In this study, simultaneous sludge reduction and nutrient removal was evaluated in laboratory scale sequencing batch reactors (SBRs). Two SBRs were operated alongside for a duration of 370d. One SBR was operated to achieve nutrient removal (control-SBR) at 10d solids retention time (SRT), while the other (modified-SBR) was operated to achieve nutrient removal along with sludge reduction. Sludge reduction in the modified-SBR was accomplished by subjecting the recycled biomass to feasting and fasting at sufficiently high SRT close to infinity (phase I and II) and finite SRT (phase III). The observed biomass yield in the modified-SBR was estimated to be 0.17mg TSSmg(-1) COD, representing 63% sludge reduction compared to the control-SBR. The NH(3) levels in the effluents from both SBRs always remained below detection limit. The average dissolved phosphorus removal efficiencies in the control-SBR and the modified-SBR were 87% and 84%, respectively, during phase II. However, the biomass of the modified-SBR increased during phase II. To control this, biomass wastage was initiated directly from the modified-SBR during phase III at a rate equivalent to the observed biomass accumulation rate in the system in phase II. This resulted in an overall 100d SRT for the modified-SBR system. Following this change, biomass accumulation in the modified-SBR was controlled, and a net 63% sludge reduction could be sustained along with 90% phosphorus and 100% NH3 removal. Consistent denitrification activities were also noticed in both SBRs despite the absence of any carbon source during the anoxic phase of every cycle.

Anaerobic ammonia oxidation (ANAMMOX) for side-stream treatment of anaerobic digester filtrate process performance and microbiology.

A laboratory scale semi‐batch fed anaerobic ammonia oxidation (ANAMMOX) reactor was operated in the lab under two different feeding operations. In the first scenario, termed as phase I, the reactor was seeded and operated with NO2‐N added externally with the filtrate to the reactor in the ratio needed for the successful ANAMMOX. A second reactor was also initiated shortly after the start‐up of the ANAMMOX to accomplish partial nitrification (nitritation reactor) to generate NO2‐N. In phase II, the operation of the ANAMMOX reactor was switched to the mode in which case the partially nitrified effluent from the nitritation reactor was fed to the ANAMMOX reactor. In both phases, real filtrate from a local wastewater treatment plant was used as the feed. The ANAMMOX reactor sustained a loading rate (average 0.33 ± 0.03 with a max of 0.4 g N (L day)−1) which is comparable with many other fed‐batch reactors in the literature. Consistent total N removal (average of 82 ± 4%) could be sustained in the ANAMMOX reactor during both phases. The nitritation reactor also consistently enabled a NO2‐N to NH3‐N ratio of 1.2:1 which was needed for the successful operation of the ANAMMOX reactor in phase II. Sequence analysis and FISH showed that Kuenenia stuttgartiensis dominated the enriched ANAMMOX community along with several unidentified, but seemingly enriched, potential ANAMMOX strains. Microbial ecology analysis for nitritation reactor showed the dominance of Nitrosomonas europaea. In summary, this manuscript provides important information on the start‐up and operation of anammox reactor with detailed investigation on microbial ecology in this reactor. Biotechnol. Bioeng. 2013; 110: 1180–1192.

Metabolic network analysis reveals microbial community interactions in anammox granules

Microbial communities mediating anaerobic ammonium oxidation (anammox) represent one of the most energy-efficient environmental biotechnologies for nitrogen removal from wastewater. However, little is known about the functional role heterotrophic bacteria play in anammox granules. Here, we use genome-centric metagenomics to recover 17 draft genomes of anammox and heterotrophic bacteria from a laboratory-scale anammox bioreactor. We combine metabolic network reconstruction with metatranscriptomics to examine the gene expression of anammox and heterotrophic bacteria and to identify their potential interactions. We find that Chlorobi-affiliated bacteria may be highly active protein degraders, catabolizing extracellular peptides while recycling nitrate to nitrite. Other heterotrophs may also contribute to scavenging of detritus and peptides produced by anammox bacteria, and potentially use alternative electron donors, such as H2, acetate and formate. Our findings improve the understanding of metabolic activities and interactions between anammox and heterotrophic bacteria and offer the first transcriptional insights on ecosystem function in anammox granules.

Effect of organic carbon on ammonia oxidizing bacteria in a mixed culture.

This study examined the effect of organic carbon (peptone vs. glucose) on two sequencing batch reactors performing simultaneous carbon oxidation and nitrification. Although each reactor had similar COD oxidation kinetics (0.029 and 0.036 mg COD mg VSS(-1) h(-1)), the Monod nitrification kinetics for the peptone-fed reactor (mu(m)=2.72 h(-1), K(s)=17.8 mg N L(-1)) were faster than for the glucose-fed reactor (mu(m)=0.868 h(-1), K(s)=26.5 mg L(-1)). The overall bacterial communities were profiled by 16S rRNA cloning and sequencing and revealed homology with a greater variety of bacteria from the peptone-fed reactor than the glucose-fed reactor. In addition, amoA cloning and sequencing, terminal restriction fragment length polymorphism, and fluorescent in situ hybridization experiments indicated greater AOB diversity and abundance in the peptone-fed reactor. This research provides evidence that the organic carbon source affects the make-up of the heterotroph community as well as AOB in mixed cultures.

Various physico-chemical stress factors cause prophage induction in Nitrosospira multiformis 25196- an ammonia oxidizing bacteria

Bacteriophages are viruses that infect bacteria and contribute significant changes in the overall bacterial community. Prophages are formed when temperate bacteriophages integrate their DNA into the bacterial chromosome during the lysogenic cycle of the phage infection to bacteria. The prophage (phage DNA integrated into bacterial genome) on the bacterial genome remains dormant, but can cause cell lysis under certain environmental conditions. This research examined the effect of various environmental stress factors on the ammonia oxidation and prophage induction in a model ammonia oxidizing bacteria Nitrosospira multiformis ATCC 25196. The factors included in the study were pH, temperature, organic carbon (COD), the presence of heavy metal in the form of chromium (VI) and the toxicity as potassium cyanide (KCN). The selected environmental factors are commonly encountered in wastewater treatment processes, where ammonia oxidizing bacteria play a pivotal role of converting ammonia into nitrite. All the factors could induce prophage from N. multiformis demonstrating that cell lysis due to prophage induction could be an important mechanism contributing to the frequent upset in ammonia oxidation efficiency in full scale treatment plants. Among the stress factors considered, pH in the acidic range was the most detrimental to the nitrification efficiency by N. multiformis. The number of virus like particles (VLPs) increased by 2.3E+10 at pH 5 in 5h under acidic pH conditions. The corresponding increases in VLPs at pH values of 7 and 8 were 9.67E+9 and 1.57E+10 in 5h respectively. Cell lysis due to stress resulting in phage induction seemed the primary reason for deteriorated ammonia oxidation by N. multiformis at lower concentrations of Cr (VI) and potassium cyanide. However, direct killing of N. multiformis due to the binding of Cr (VI) and potassium cyanide with cell protein as demonstrated in the literature at higher concentrations of these toxic compounds was the primary mechanism of cell lysis of N. multiformis. Organics represented by the chemical oxygen demand (COD) did not have any effect on the phage induction in N. multiformis. This AOB remained dormant at low temperature (4 degrees C) without any phage induction. Significant decrease in the number of live N. multiformis cells with a corresponding increase in the number of VLPs was recorded when the temperature was increased to 35 degrees C. Death of N. multiformis at 45 degrees C was attributed to the destruction of cell wall rather than to the phage induction.

Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibiotic‐resistant bacterial biofilms

To demonstrate elimination of bacterial biofilm on membranes to represent wastewater treatment as well as biofilm formed by antibiotic‐resistant bacterial (ARB) to signify medical application, an antibiotic‐resistant bacterium and its lytic bacteriophage were isolated from a full‐scale wastewater treatment plant. Based on gram staining and complete 16 S rDNA sequencing, the isolated bacterium showed a more than 99% homology with Delftia tsuruhatensis, a gram‐negative bacterium belonging to β‐proteobacteria. The Delftia lytic phages draft genome revealed the phage to be an N4‐like phage with 59.7% G + C content. No transfer RNAs were detected for the phage suggesting that the phage is highly adapted to its host Delftia tsuruhatensis ARB‐1 with regard to codon usage, and does not require additional tRNAs of its own. The gene annotation of the Delftia lytic phage found three different components of RNA polymerase (RNAP) in the genome, which is a typical characteristic of N4‐like phages. The lytic phage specific to D. tsuruhatensis ARB‐1 could successfully remove the biofilm formed by it on a glass slide. The water flux through the membrane of a prototype lab‐scale membrane bioreactor decreased from 47 L/h m2 to ∼15 L/h m2 over 4 days due to a biofilm formed by D. tsuruhatensis ARB‐1. However, the flux increased to 70% of the original after the lytic phage application. Overall, this research demonstrated phage therapys great potential to solve the problem of membrane biofouling, as well as the problems posed by pathogenic biofilms in external wounds and on medical instruments. Biotechnol. Bioeng. 2015;112: 1644–1654.

Biofilm control with natural and genetically-modified phages.

Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.