Impact of the addition of a nitrifying activated sludge on ammonia oxidation during composting of residual household wastes.

AIMS: To investigate the nitrogen-microbial community dynamics during composting of a mixture of nitrifying waste activated sludge (WAS) and fine organic fraction of residual household waste (RHW). To examine whether the addition of nitrifying sludge could promote ammonia oxidation and reduce ammonia emissions. METHODS AND RESULTS: The fine organic fraction of RHW was mixed with the WAS and homogenized. The mixture and each waste alone were loaded in aerobic cells under controlled conditions, respectively. Both nitrogen and microbial community dynamics were monitored during 50 days of composting. The ammonia oxidizers were quantified and identified in the sludge and compost. The changes in ammonia-oxidizing bacteria (AOB) concentrations corresponded to the ammonia oxidation rates calculated from nitrogen balance. The addition of WAS did not efficiently reduce ammonia emissions because the Nitrosomonas oligotropha-like AOB introduced declined during the active stage of composting. Ammonia oxidation was probably limited by the intense heterotrophic activities at the active stage. Nitrosomonas europaea/eutropha and Nitrosomonas nitrosa-like AOB were established only during the maturation stage. They were the main contributors to ammonia oxidation during composting. CONCLUSIONS: The mixing of nitrifying WAS with the RHW during the early stages of composting does not promote ammonia oxidation nor reduce ammonia emissions because of limiting biologic factors during the active stage of composting. SIGNIFICANCE AND IMPACT OF THE STUDY: The mixing of activated sludge with RHW before composting is a common practice on composting plants. This study proved the limitation of this practice to reduce ammonia emissions during composting via bioaugmentation of ammonia-oxidizing organisms. It correlated successfully the ammonia oxidation rate with different groups of ammonia oxidizers and explains the fail of promoting ammonia oxidation during the early stages of composting. It suggests Nit. europaea/eutropha and Nit. nitrosa-like AOB were the main contributors to ammonia oxidation during composting.

Effect of land application of manure from enrofloxacin-treated chickens on ciprofloxacin resistance of Enterobacteriaceae in soil.

A field plot experiment was carried out to evaluate the impact of spreading chicken manure containing enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP), on the levels of CIP-resistant Enterobacteriaceae in soil. The manures from chickens treated with ENR and from untreated control chickens were applied on six plots. Total and CIP-resistant Enterobacteriaceae were counted on Violet Red Bile Glucose medium containing 0 to 16mg L(-1) of CIP. A total of 145 isolates were genotyped by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The minimum inhibitory concentration (MIC) of CIP for the isolates of each ERIC-PCR profile was determined. The most frequently isolated Enterobacteriaceae included Escherichia coli, and to a lesser extent, Enterobacter and 5 other genera from environmental origin. The composition of the E. coli community differed between manure and manured soil suggesting that the E. coli genotypes determined by ERIC-PCR varied significantly in their ability to survive in soil. One of these genotypes, including both susceptible and resistant isolates, was detected up to 89 days after the manure was applied. Most of the E. coli isolated in soil amended with manure from treated chickens was resistant to CIP (with a MIC ranging between 2 and 32mg L(-1)). In contrast, despite the presence of ENR in soil at concentrations ranging from 13-518µg kg(-1), the environmental Enterobacteriaceae isolates had a CIP MIC≤0.064mg L(-1), except one isolate which had a MIC of 0.25mg L(-1), These results showed that spreading manure from ENR-treated chickens enabled CIP-resistant E. coli to persist for at least three months in the soil. However, neither the presence of fluoroquinolones, nor the persistence of CIP-resistant E. coli, increased the CIP-susceptibility of environmental Enterobacteriaceae.

Sulphur fate and anaerobic biodegradation potential during co-digestion of seaweed biomass (Ulva sp.) with pig slurry.

Seaweed (Ulva sp.) stranded on beaches were utilized as co-substrate for anaerobic digestion of pig slurry in three-month co-digestion tests in pilot scale anaerobic digesters in the laboratory. The methanogenic potential of Ulva sp. was low compared to that of other potential co-substrates available for use by farmers: 148 N m3CH4/t of volatile solids or 19 N m3CH4/t of crude product. When used as a co-substrate with pig manure (48%/52% w/w), Ulva sp. seaweed did not notably disrupt the process of digestion; however, after pilot stabilisation, biogas produced contained 3.5% H2S, making it unsuitable for energy recovery without treatment. Sequentially addition of the sulphate reduction inhibitor, potassium molybdate, to a final concentration of 3mM, temporarily reduced H2S emissions, but was unable to sustain this reduction over the three-month period. According to these pilot tests, the use of seaweed stranded on beaches as co-substrate in farm-based biogas plants shows some limitations.

Technical and economical assessment of formic acid to recycle phosphorus from pig slurry by a combined acidification-precipitation process.

Dissolution by acidification followed by a liquid/solid separation and precipitation of phosphorus from the liquid phase is one possibility to recycle phosphorus from livestock effluents. To avoid increase of effluent salinity by using mineral acids in the recycling process, the efficiency of two organic acids, formic and acetic acid, in dissolving the mineral phosphorus from piggery wastewater was compared. The amount of formic acid needed to dissolve the phosphorus was reduced three fold, compared to acetic acid. The amount of magnesium oxide needed for further precipitation was decreased by two with formic acid. Neither the carbon load nor the effluent salinity was significantly increased by using formic acid. An economical comparison was performed for the chemical recycling process (mineral fertilizer) vs. centrifugation (organic fertilizer) considering the centrifugation and the mineral fertilizers sold in the market. After optimisation of the process, the product could be economically competitive with mineral fertilizer as superphosphate in less than 10 years.

A Saccharomyces cerevisiae-based bioassay for assessing pesticide toxicity.

This study evaluates the toxic effect of three pesticides (Azoxystrobin, Cymoxanil, and Diuron) on the yeast Saccharomyces cerevisiae for the development of a new bioassay based on inhibition of S. cerevisiae metabolic activity at the level of adenosine-5-triphosphate (ATP) synthesis, as compared with two different toxicity tests based on inhibition of Daphnia magna mobility (NF EN ISO 6341) and inhibition of Vibrio fisheri activity (NF EN ISO 11348). The S. cerevisiae bioassay is cheaper and 96 times faster than the D. magna toxicity bioassay, but has lower sensitivity. It is as fast as the V. fisheri bioassay and more sensitive. Thus, this new toxicity test can be proposed for rapid detection of pesticide residues in environmental samples as a complement to the more expensive and time-consuming D. magna toxicity test.

Microbiology and performance of a methanogenic biofilm reactor during the start-up period.

AIMS: To understand the interactions between anaerobic biofilm development and process performances during the start-up period of methanogenic biofilm reactor. METHODS AND RESULTS: Two methanogenic inverse turbulent bed reactors have been started and monitored for 81 days. Biofilm development (adhesion, growth, population dynamic) and characteristics (biodiversity, structure) were investigated using molecular tools (PCR-SSCP, FISH-CSLM). Identification of the dominant populations, in relation to process performances and to the present knowledge of their metabolic activities, was used to propose a global scheme of the degradation routes involved. The inoculum, which determines the microbial species present in the biofilm influences bioreactor performances during the start-up period. FISH observations revealed a homogeneous distribution of the Archaea and bacterial populations inside the biofilm. CONCLUSION: This study points out the link between biodiversity, functional stability and methanogenic process performances during start-up of anaerobic biofilm reactor. It shows that inoculum and substrate composition greatly influence biodiversity, physiology and structure of the biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of molecular techniques associated to a biochemical engineering approach is useful to get relevant information on the microbiology of a methanogenic growing biofilm, in relation with the start-up of the process.

The efficiency of biological aerobic treatment of piggery wastewater to control nitrogen, phosphorus, pathogen and gas emissions.

Due to the water pollution and in order to reduce the nitrogen load applied on soils, biological nitrogen removal treatment of piggery wastewaters was developed in Brittany (France), with 250-300 units running. Four types of treatment processes were built including a biological reactor allowing to remove about 60-70% of the nitrogen content as gas by nitrification/denitrification. The addition of different mechanical separators (screw-press, centrifuge decanter ...) led to concentration of phosphorus in an exportable solid phase, allowing a reduction up to 80% of the phosphorus applied locally on soils. Moreover, a reduction of the gaseous emissions was observed using this management process as compared to conventional management (storage + land spreading) including ammonia (up to 68%) and greenhouse gases (55%). Finally, the level of enteric and pathogenic bacteria was also decreased with the treatment process as compared to conventional management systems. However, in spite of these results, the significant cost of the treatment must be underlined and alternative systems including anaerobic digestion will have to be studied.

The effect of incubation conditions on the laboratory measurement of the methane producing capacity of livestock wastes.

The effects of incubations conditions (dilution, mixing, incubation time and inoculum amount and origin) on the determination of the maximum methane producing capacity (B(0)) from various livestock slurries were evaluated. For this purpose, the methane yields of different livestock slurries were determined using batch anaerobic incubations performed at 30 degrees C as regard these different conditions. The B(0) and the methane (CH(4)) generation as a function of time were used to study the processes and to determine the best incubation conditions. Methanogenesis was identified as the major rate-limiting step during the anaerobic degradation of slurries, probably due to inhibition by volatile fatty acids. In some cases, high free NH(3) concentrations were suspected to inhibit the hydrolysis process. The addition of inoculum and/or the dilution of the substrate reduced the inhibition and allowed to reach the B(0) more rapidly. However, the addition of inoculum must be minimized to reduce the possible errors made by considering a similar production by the inoculum with and without the substrate. All experiments performed during this study allowed to define the incubation conditions required for the determination of the B(0) from livestock slurries. Applying these conditions, the B(0) values determined for swine slurries varied from 244 to 343L CH(4)kg V(added)(-1), from 204 to 296L CH(4)kg V(added)(-1) for dairy cattle slurries and equalled 386 and 319L CH(4)kg VS(added)(-1) respectively for calves and duck slurries.

Influence of closed loop control on microbial diversity in a nitrification process.

This paper compares two control strategies for a nitrification process. The objective is to achieve partial nitrification and thus to accumulate nitrite instead of nitrate. To this end, change in temperature setpoint and active control of oxygen and ammonia concentrations are evaluated in the long term. Evaluation is made on the control performances that are obtained, but also--and more importantly--on the microbial diversity. In particular, it is shown that the combined oxygen and ammonia control strategy is more appropriate since shift in the temperature setpoint strongly affects the composition of the microbial ecosystem present in the reactor whereas active control of oxygen and ammonia does not.

The microbial signature of drinking waters: myth or reality?

This paper presents a new software developed for analyzing single strand conformation polymorphism (SSCP) electrophoresis patterns delivered by the genetic analyzer ABI310 (Applied Biosystems). SSCP is a molecular typing technique based on the PCR amplification of microbial 16S rDNA and used for the monitoring of complex microbial ecosystems dynamics. The software--a home-made MATLAB toolbox called MODIMECO--developed for the analysis of SSCP patterns is presented. MODIMECO includes a number of basic signal processing abilities as well as largely used statistical tools such as the well known principal component analysis. The use of the SSCP for assessing the hypothesis of the existence of a microbial signature of drinking waters illustrates the typical advantages of using such software tools. Results are discussed and conclusions drawn.

Effect of solid hold-up on nitrite accumulation in a biofilm reactor--molecular characterization of nitrifying communities.

Biological ammonium oxidation was carried out in two inverse turbulent bed reactors fed with synthetic mineral wastewater containing a high ammonium concentration (100 mg N-NH4+/L). Both reactors were started-up and operated in the same conditions except for the solid carrier concentration: the solid hold-up ratios applied, defined as the ratios of static to expanded bed height, were 0.1 and 0.3 in reactors R10 and R30 respectively. These two solid hold-up ratios generate different particle-to-particle collision frequencies and, therefore, detachment forces. The influence of solid hold-up on biofilm growth and nitrifying performance was studied from a macroscopic (i.e. nitrate and/or nitrite production) and microbiological point of view. After 60 days of operation, both reactors contained the same amount of biomass. However, R10 produced only nitrate while nitrite accumulated in R30. A comparison of microbial populations in the reactors showed that R10 contained both ammonium and nitrite oxidizing populations such as Nitrosomonas and Nitrospira, whereas in R30, ammonium oxidizing populations were much greater than those of nitrite oxidizers. The major ammonium-oxidizing organism was not the same in both reactors.

16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure.

A laboratory-scale continuously stirred anaerobic thermophilic batch digester was inoculated with cattle manure. Bacterial and archaeal communities, as well as digester performances, were analysed during reactor start-up for about 20 days. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used for overall detection and for study of the dynamics of microbial populations. Dominant bacteria and archaea 16S rDNAs were sequenced from the sample on day 12. Ten bacteria and 3 archaea OTUs (operational taxonomic units) were identified from the 52 clones sequenced. Sequences corresponding to the dominant bacterial SSCP peak were phylogenetically close to the 16S rDNA sequence of Bacillus thermoterrestris, whereas sequences corresponding to the two dominant archaeal SSCP peaks were phylogenetically close to the 16S rDNA sequence of Methanoculleus thermophilicus and Methanosarcina thermophila.

Evolution of the bacterial community during granules formation in denitrifying reactors followed by molecular, culture-independent techniques.

The microbial community in two acetate-fed denitrifying reactors, inoculated with methanogenic sludge, was monitored by 16S rDNA-based methods (SSCP and FISH). Both reactors converged to similar, stable communities. The predominant organisms belonged to the genera Thauera, Paracoccus and Denitrobacter, detected both by molecular and culture-based methods.

Community analysis of a denitrifying reactor treating landfill leachate.

The bacterial community of a denitrifying reactor from a system for landfill leachate decontamination was studied applying cultivation methods, denitrifying activity measurements and characterisation of community 16S rDNA. The sludge presented a high denitrifying activity but a relatively low number of denitrifying bacteria as determined by most probable number. Over 50% of the sequences retrieved in the molecular analysis were related to genera with the capacity to denitrify in the alpha- and beta-subdivisions of the Proteobacteria. Fifteen percent of the DNAs were related to not yet cultured organisms belonging to the green non-sulphur phylum. High similarity values between sequences from isolates and clones were observed.

Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration.

The microbial community of a conventional anaerobic-aerobic sequencing batch reactor was investigated by cloning and sequencing bacterial 16S rDNA. The 92 16S rDNA sequences analysed ranged across 50 different operational taxonomic units (OTU). The majority of these sequences were not closely related to known species. They belonged to 12 different groups, but essentially to the Cytophagales and the Proteobacteria beta, which represented 38% and 17% of the retrieved sequences respectively. No OTU numerically outnumbered the others. However, similarities were observed with previous reports on molecular characterisation of phosphorus-accumulating ecosystems, suggesting an enrichment in microorganisms belonging to the Rhodocyclus group. Thereafter, the ability of this anaerobic-aerobic microbial community to accumulate phosphorus with nitrate as its energy source was investigated. The reactor was shifted from anaerobic-aerobic running conditions to anaerobic-anoxic conditions by injection of nitrate; and its microbial community was monitored by PCR-single strand conformation polymorphism (SSCP). The reactor maintained a good phosphorus accumulation and similar SSCP microbial community patterns for a period of 17 days, suggesting that the same microbial community was able to respire both oxygen and nitrate. However, this situation was unstable, since a breakdown in phosphorus accumulation occurred thereafter.

Comamonas nitrativorans sp. nov., a novel denitrifier isolated from a denitrifying reactor treating landfill leachate.

A group of Gram-negative denitrifying bacteria has been isolated from a denitrifying reactor treating landfill leachate. The new isolates produced both oxidase and catalase and showed growth on acetate, butyrate, n-caproate, i-butyrate, i-valerate, propionate, n-valerate, lactate, alanine, benzoate, phenylalanine and ethanol. No growth was observed on sugars. The bacteria could perform anoxic reduction of nitrate, nitrite and nitrous oxide to nitrogen, coupled to the oxidation of the same substrates as those used under aerobic conditions, except for aromatic compounds. They were very efficient denitrifiers, as estimated from the specific rate of N2 gas production. All the strains showed the same 16S rDNA restriction profile and one of them, designated 23310T, was selected for phylogenetic analysis. The organism clustered within the family Comamonadaceae, being related to Comamonas terrigena (95.8% sequence similarity). On the basis of the phylogenetic analysis, physiological characterization and the ability to efficiently reduce nitrate to N2, it is proposed that the bacterium be assigned to a new species, Comamonas nitrativorans. The type strain is 23310T (= DSM 13191T = NCCB 100007T = CCT 7062T).

Monitoring of the microbial community of a sequencing batch reactor bioaugmented to improve its phosphorus removal capabilities.

The acclimatisation of an activated sludge to enhanced biological phosphorus removal conditions was followed after and without bioaugmentation with a low amount of phosphorus-accumulating sludge. Phosphorus removal yields were monitored by conventional analytical methods and microbial communities evolutions were followed by a finger printing molecular technique (PCR-SSCP). While the benefit of the bioaugmentation seems real at the level of the reactor parameters, bioaugmentation speeded up the installation of good and stable phosphorus removal yield, the establishment of the inoculated microbial community in the bioaugmented reactor is still unclear. Both the bioaugmented and the control microbial communities evolved in a similar way to end up with apparently comparable populations. At the time of the experiment, the results suggest that the microbial community inoculated for the bioaugmentation did not establish in the reactor but compensated for phosphorus accumulation until the acclimatisation of an endogenous microbial community arose.

Evaluation of the denitrifying microbiota of anoxic reactors.

Removal of inorganic nitrogen compounds from wastewaters can be accomplished by a combination of the biological processes of nitrification and denitrification. The information on the microbiota present in denitrifying reactors is still scarce. In the present work the evaluation of the denitrifying microbiota of different reactor sludges was performed by specific activity measurements and MPN count of denitrifiers. We also present the isolation and physiological and phylogenetic characterisation of denitrifying bacteria from the anoxic reactor of a combined system treating landfill leachate. Specific denitrifying activity measurements were faster to perform and more reliable than MPN enumerations. 16S rDNA characterisation of the isolates showed that they belonged to the genera Thauera, Acidovorax and Alcaligenes and were closely related to microorganisms retrieved from ecosystems rich in recalcitrant compounds. Two of the isolates could grow on aromatic compounds as sole carbon source.

Ecological study of a bioaugmentation failure.

A nitrifying sequencing batch reactor was inoculated twice with the aerobic denitrifying bacterium Microvirgula aerodenitrificans and fed with acetate. No improvement was obtained on nitrogen removal. The second more massive inoculation was even followed by a nitrification breakdown, while at the same time, nitrification remained stable in a second reactor operated under the same conditions without bioaugmentation. Fluorescent in situ hybridization with rRNA-targeted probes revealed that the added bacteria almost disappeared from the reactor within 2 days, and that digestive vacuoles of protozoa gave strong hybridization signals with the M. aerodenitrificans-specific probe. An overgrowth of protozoa, coincident with the disappearance of free-living bacteria, was monitored by radioactive dot-blot hybridization only in the bioaugmented reactor. Population dynamics were analysed with a newly developed in situ quantification procedure of the probe-targeted bacteria. The nitrifying groups of bacteria decreased in a similar way in the bioaugmented and non-bioaugmented reactors. Other bacterial groups evolved differently. The involvement of different ecological parameters are discussed separately for each reactor. These results underline the importance of predator-prey interaction and illustrate the undesirable effects of massive bioaugmentation.

Gene replacement with linear DNA in electroporated wild-type Escherichia coli.

Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA. Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.