Biotechnological conversion of agro-industrial wastewaters into biodegradable plastic, poly beta-hydroxybutyrate

Waste activated sludge generated from a combined dairy and food processing industry wastewater treatment plant was evaluated for its potential to produce biodegradable plastic, poly beta-hydroxybutyric acid (PHB). Deproteinized jowar grain-based distillery spentwash yielded 42.3% PHB production (w/w), followed by filtered rice grain-based distillery spentwash (40% PHB) when used as substrates. Addition of di-ammonium hydrogen phosphate (DAHP) resulted in an increase in PHB production to 67% when raw rice grain-based spentwash was used. Same wastewater, after removal of suspended solids by filtration and with DAHP supplementation resulted in lower PHB production (57.9%). However, supplementing other wastes with DAHP led to a substantial decrease in PHB content in comparison to what was observed in the absence of DAHP.     

Microbial Ecology of Activated Sludge: II. Bacteriophages, Bdellovibrio, Coliforms, and Other Organisms

A comparative estimation of the coliform population of raw sewage, activated sludge, and the effluent derived therefrom revealed that raw sewage had a preponderance of Escherichia coli (75%), as compared with 25 and 30%, respectively, in sludge and effluent. Nitrogen-free mannitol-sucrose enrichments of activated sludge resulted in the isolation of Azotobacter agilis, Aerobacter aerogenes, Corynebacterium laevaniformans, and an Achromabacter species. Sludge had a large population of C. laevaniformans and A. aerogenes but not of Azotobacter. The bacterial parasites, Bdellovibrio and bacteriophages, were not active during activated-sludge treatment. A 10-fold reduction in phage content occurred after 2 hr of aeration, but the Bdellovibrio population was unaffected.     

Performance of a commercial inoculum for the aerobic biodegradation of a high fat content dairy wastewater

The effectiveness of a commercial inoculum for degrading a dairy wastewater with high fat content was evaluated, and compared with an activated sludge inoculum from a dairy wastewater treatment pond. Both inocula reached similar chemical oxygen demand removal in batch experiments. The population dynamics was also studied by determining heterotrophic counts. Predominant microorganisms were differentiated by colony morphology and genomic fingerprinting (BOX-PCR) analysis. The higher population diversity and the wider range of CO2 production rate observed in batch reactors inoculated with activated-sludge, indicated that microorganisms from this inoculum were well adapted and may have had synergic activity for the degradation of the dairy effluent. When the bioreactor was operated with the commercial inoculum in continuous mode, according to its microbial growth kinetics, other microorganisms became predominant. These results showed that inoculated microorganisms did not persist in the open system and periodic addition of microorganisms may be needed to achieve a high performance treatment.     

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

The importance of having a rapid method for determining the viable biomass in activated-sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out-of-coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated-sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed-liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

One-year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated-sludge purification plant.

Samples of raw sewage, primary effluent, and secondary effluent from a large activated-sludge purification plant near Melbourne (Victoria, Australia) were collected every second week for 1 year. Viruses were detected in all secondary effluent samples and in six of seven samples obtained after final chlorination. Adenoviruses (85% reduction) and reoviruses (28% reduction) were removed less efficiently by this treatment process than were enteroviruses (93% reduction). In addition, 57 of 171 samples of effluent tested were positive for either adenoviruses or reoviruses, or both, when enteroviruses were not isolated. This clearly shows that the use of enteroviruses as sole indicators of viruses in water may miss up to one-third of instances of viral contamination. Enteroviruses and adenoviruses were isolated most frequently in HeLa-R cell cultures, whereas reoviruses were most often isolated in primary monkey kidney cells.     

Efficacy of activated sludge in removing Cryptosporidium parvum oocysts from sewage.

Primary clarifier effluent (procedure B) and final effluent (procedure A) from a wastewater treatment plant were enriched with Cryptosporidium parvum oocysts obtained from the feces of naturally infected calves. Procedure B samples alone were subjected to a laboratory simulation of activated-sludge treatment. Coccidium-free neonatal CD-1 mice were then inoculated intragastrically with procedure A or procedure B samples. Seven days after inoculation, the intensity of oocyst infection in procedure B mice was 91% less than in procedure A mice (controls).     

Efficacy of activated sludge in removing Cryptosporidium parvum oocysts from sewage.

Primary clarifier effluent (procedure B) and final effluent (procedure A) from a wastewater treatment plant were enriched with Cryptosporidium parvum oocysts obtained from the feces of naturally infected calves. Procedure B samples alone were subjected to a laboratory simulation of activated-sludge treatment. Coccidium-free neonatal CD-1 mice were then inoculated intragastrically with procedure A or procedure B samples. Seven days after inoculation, the intensity of oocyst infection in procedure B mice was 91% less than in procedure A mice (controls).     

Assessment of physiological state of microorganisms in activated sludge with flow cytometry: application for monitoring sludge production minimization

Many sludge reduction processes have been studied for the minimization of sludge production in biological wastewater treatment. The investigations on most of these processes have monitored the increase of the soluble chemical oxygen demand, the sludge mass reduction, or the decrease of the floc size, but little information has been obtained on cell lysis and the change of the biological cell activity. However, employing any strategy for reducing sludge production may have an impact of microbial community in biological wastewater treatment process. This impact may influence the sludge characteristics and the quality of effluent. The objective of this study concerns the determination of the physiological state of activated sludge microorganisms during a sludge minimization process. A thermal treatment at 80 °C for 5, 20, 40 and 60 min was chosen in this study. Staining bacteria with CTC and SYTOX green was used to evaluate biological cell activity and viability of cell types contained in activated sludge, respectively. The monitoring of cell activity and viability was performed using flow cytometry (FCM) analysis before and after thermal treatment of activated sludge. Results indicated an increase in the number of permeabilized cells and a decrease in the number of active cells, subsequent to the thermal treatment. The study also confirms the potential of FCM to successfully evaluate the physiological heterogeneity of an activated sludge bacterial population. Moreover, the experimentally observed correlations between the FCM results and the organic matter solubilization in activated sludge samples during thermal treatment revealed that the increase in the soluble organic matter concentration was predominantly due to an intracellular material release. Identifying the increase in activated sludge hydrolysis requires a precise knowledge of the involved mechanisms, and this study indicated that the FCM, used in conjunction with specific probes, could be a useful tool.     

Viruses in sewage: effect of phosphate removal with calcium hydroxide (lime).

During calcium hydroxide (lime) treatment (pH 9.6 to 10.5) of wastewaters for phosphate removal there was also a two-log removal of added poliovirus (type I, Sabin) from effluents. A similar virus reduction was seen in the sludge generated in these experiments. However, in view of the limitations of techniques for virus recovery from sludge, only a small portion of the infectious virus present in lime sludge may have been detected. Storage of lime sludge at 28 degrees C for up to 48 h produced no appreciable reduction in the virus titre. Five sets of field samples of sewage, effluents, and sludge from a sewage treatment plant (Kemptville, Ont.) which utilizes lime for phosphate removal were also examined for indigenous viruses being BS-C-1 cells. All of the sample of lime sludge and 80% of the samples of both sewage and lime-treated effluent revealed virus; after chlorination only 20% of the lime-treated effluent samples were positive for virus. In contrast, in an earlier study with essentially the same experimental set up, 76% of the sample of chlorinated primary effluent were found to contain virus. Because of the easily detectable quantities of infectious virus in lime sludge and due to the lack of virus inactivation during storage of such sludge, caution must be exercised in its handling and disposal.     

Excess sludge reduction by biological way: from experimental experience to a real full scale application

The aim of this study was to investigate the ability of a biological process applied in the sludge line and based on the alternation of oxic and anoxic phases, to minimize the waste sludge production. After some tests in pilot scale, the process was applied in a real municipal wastewater treatment plant of 35,000 PE trying out one setting of working experimental conditions. A rate of the recycle flow was conditioned in a treatment basin, maintaining an HRT of 10 days. The control device operated for the 50% of time in the ORP range between -400 and -200 mV, and for the 50% in the ORP range between -200 and +50 mV. The mass balance defined an actual observed growth yield equal to 0.09 kgTVSkgCODr(-1), and the heterotrophic yield values, assessed by batch tests, seemed to be a suitable marker for the sludge reduction and for the energy uncoupling.     

Use of protozoa and metazoa for decreasing sludge production in aerobic wastewater treatment

Summary A new approach to decreasing sludge production in aerobic biological wastewater treatment involving use of protozoa and metazoa was tested. The dissolved organics in the two synthetic wastewaters (based on acetic acid and methanol, respectively) tested were decomposed to >90% and the biomass production was decreased by 60–80%. The total sludge yield, expressed as total suspended solids per gram chemical oxygen demand removed, was 0.17 g TSS/g COD in the system fed acetic acid, whereas it was 0.05 g TSS/g COD in the system fed methanol. The explanation for this difference was that in the system fed methanol, dispersed bacteria were obtained that were easily grazed by the protozoa and metazoa in the predator stage. In the system fed acetic acid, the bacteria formed zoogloeal flocs, which protected them from grazing in the predator stage. With both carbon sources a significant release of nitrate (> 7 mg N/l) and of phosphate (> 2.5 mg P/l) was observed in the effluent.     

Energy use and recovery strategies within wastewater treatment and sludge handling at pulp and paper mills

This paper presents an inclusive approach with focus on energy use and recovery in wastewater management, including wastewater treatment (WWT) and sludge handling. Process data from three Swedish mills and a mathematical model were used to evaluate seven sludge handling strategies. The results indicate that excess energy use in WWT processes counters the potential energy recovery in the sludge handling systems. Energy use in WWT processes is recommended to aim for sufficient effluent treatment, not for sludge reduction. Increased secondary sludge production is favourable from an energy point of view provided it is used as a substrate for heat, biogas or electricity production.     

Sludge minimisation technologies

The treatment and disposal of excess sludge represents a bottleneck of wastewater treatment plants all over the world, due to environmental, economic, social and legal factors. There is therefore a growing interest in developing technologies to reduce the wastewater sludge generation. The goal of this paper is to present the state-of-the-art of current minimisation techniques for reducing sludge production in biological wastewater treatment processes. An overview of the main technologies is given considering three different strategies: The first option is to reduce the production of sludge by introducing in the wastewater treatment stage additional stages with a lower cellular yield coefficient compared to the one corresponding to the activated sludge process (lysis-cryptic growth, uncoupling and maintenance metabolism, predation on bacteria, anaerobic treatment). The second choice is to act on the sludge stage. As anaerobic digestion is the main process in sewage sludge treatment for reducing and stabilising the organic solids, two possibilities can be considered: introducing a pre-treatment process before the anaerobic reaction (physical, chemical or biological pre-treatments), or modifying the digestion configuration (two-stage and temperature-phased anaerobic digestion, anoxic gas flotation). And, finally, the last minimisation strategy is the removal of the sludge generated in the activated sludge plant (incineration, gasification, pyrolysis, wet air oxidation, supercritical water oxidation).     

Effect of 3,3',4',5-tetrachlorosalicylanilide on reduction of excess sludge and nitrogen removal in biological wastewater treatment process

A metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), was used to reduce excess sludge production in biological wastewater treatment processes. Batch experiments confirmed that 0.4 mg/l of TCS reduced the aerobic growth yield of activated sludge by over 60%. However, the growth yield remained virtually constant even at the increased concentrations of TCS when cultivations were carried out under the anoxic condition. Reduction of sludge production yield was confirmed in a laboratory-scale anoxic-oxic process operated for 6 months. However, it was found that ammonia oxidation efficiency was reduced by as much as 77% in the presence of 0.8 mg/l of TCS in the batch culture. Similar results were also obtained through batch inhibition tests with activated sludges and by bioluminescence assays using a recombinant Nitrosomonas europaea (pMJ217). Because of this inhibitory effect of TCS on nitrification, the TCS-fed continuous system failed to remove ammonia in the influent. When TCS feeding was stopped, the nitrification yield of the process was resumed. Therefore, it seems to be necessary to assess the nitrogen content of wastewater if TCS is used for reducing sludge generation.     

Detection of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in effluents and sludge of a hospital sewage treatment plant.

AIMS: To detect ESBL (extended-spectrum beta-lactamase)-producing Klebsiella pneumoniae present in the effluents and sludge of a hospital sewage treatment plant, evaluating the treatment plant's potential to remove these micro-organisms. METHODS AND RESULTS: Twenty samples (crude sewage, UASB reactor effluent, filtered effluent and sludge) were collected in the period from May to December 2006, in order to analyse antimicrobial susceptibility and to check ESBL production, the disc-diffusion and the combined disc methods were used. Total and faecal coliform concentrations were also determined. ESBL-producing K. pneumoniae were detected in all samples analysed, representing 46 x 5% of the total strains isolated. Among the non-ESBL-producing strains, 26% were multiresistant and one strain resistant to eight of the nine antimicrobials tested was detected in the treated effluent. CONCLUSIONS: The hospital wastewater treatment plant did not show a satisfactory efficacy in removing pathogenic micro-organisms, allowing for the dissemination of multiresistant bacteria into the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The inefficacy of hospital wastewater treatment plants can result in routes of dissemination of multiresistant bacteria and their genes of resistance into the environment, thus contaminating water resources, and having serious negative impact on public health.     

Production of biodegradable plastics from activated sludge generated from a food processing industrial wastewater treatment plant

Most of the excess sludge from a wastewater treatment plant (60%) is disposed by landfill. As a resource utilization of excess sludge, the production of biodegradable plastics using the sludge has been proposed. Storage polymers in bacterial cells can be extracted and used as biodegradable plastics. However, widespread applications have been limited by high production cost. In the present study, activated sludge bacteria in a conventional wastewater treatment system were induced, by controlling the carbon: nitrogen ratio to accumulate storage polymers. Polymer yield increased to a maximum 33% of biomass (w/w) when the C/N ratio was increased from 24 to 144, where as specific growth yield decreased with increasing C/N ratio. The conditions which are required for the maximum polymer accumulation were optimized and are discussed.     

Anaerobic filters for the treatment of coal gasification wastewater

A process train consisting of the following sequence of unit processes, a berl-saddle-packed anaerobic filter, an expanded bed, granular activated carbon anaerobic filter, and an activated sludge nitrification system was evaluated for the treatment of a synthetically prepared coal gasification wastewater. The first-stage anaerobic filter resulted in very little removal of organic matter and no methane production. Excellent reduction in organic matter occurred in the granular activated carbon anaerobic filter. The removal mechanism was initially adsorptive and near the end of the study, removal of organic matter was primarily through conversion to methane gas. It is felt that the success of the activated carbon anaerobic filter was due to the ability of the activated carbon to sequester some components of the wastewater that were toxic to the mixed culture of anaerobic microorganisms. The activated sludge nitrification system resulted in complete ammonia oxidation and was very efficient in final effluent polishing.     

Relationship of species-specific filament levels to filamentous bulking in activated sludge

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.     

Microbial community origin and fate through a rural wastewater treatment plant

Conventional wastewater treatment relies on a complex microbiota; however, much of this community is still to be characterized. To better understand the origin, dynamics and fate of bacteria within a wastewater treatment plant: untreated primary wastewater, activated sludge and post-treatment effluent were characterized. From 3163 exact sequence variants (ESVs), 860 were annotated to species-level. In primary wastewater, 28% of ESVs were putative bacterial species previously associated with humans, 14% with animals and 5% as common to the environment. Differential abundance analysis revealed significant relative reductions in ESVs from potentially human-associated species from primary wastewater to activated sludge, and significant increases in ESVs from species associated with nutrient cycling. Between primary wastewater and effluent, 51% of ESVs from human-associated species did not significantly differ, and species such as Bacteroides massiliensis and Bacteroides dorei increased. These findings illustrate that activated sludge increased extracellular protease and urease-producing species, ammonia and nitrite oxidizers, denitrifiers and specific phosphorus accumulators. Although many human-associated species declined, some persisted in effluent, including strains of potential health or environmental concern. Species-level microbial assessment may be useful for understanding variation in wastewater treatment efficiency as well as for monitoring the release of microbes into surface water and the wider ecosystem.