Hyperthermophilic hydrogen production in a simplified reaction medium containing onion wastes as a source of carbon and sulfur.

In this study, the hyperthermophilic dark fermentation of onion wastes (OW) for hydrogen production was investigated. OW were used at different proportions in mixed fruit and vegetable wastes (FVW) to evaluate their effect on hydrogen production by Thermotoga maritima. Fermentations were performed in a pH-controlled batch stirred tank reactor (BSTR) using seawater as a simplified reaction medium. Results showed that increasing OW proportions in total fruit and vegetable wastes (tFVW) improved H2 production. Therefore, increasing the OW to tFVW ratio from 0 to 0.8 increased the cumulative H2 production from 109 to 223.6 mmol/L. The H2 productivity was also improved from 7.3 to 28.82 mmol/h.L. In fact, OW contain carbohydrates, sulfur compounds, and other nutrients, which were used as a carbon source and energetic substrate for H2 production by the halophilic bacterium T. maritima in seawater without additional chemical compounds. Then, a H2 yield of 3.36 mol H2/mol hexose was achieved using 200 mL of OW, containing 55 mmol/L of carbohydrates. A concept of H2 production from FVW at high proportions of OW in a simplified reaction medium was proposed. It allowed a H2 yield of 209 LH2/kg volatile solids which could be an interesting future alternative to the current fossil fuel.

Reduction of adsorbed dyes content in the discharged sludge coming from an industrial textile wastewater treatment plant using aerobic activated sludge process.

Dye mass balance study at full-scale industrial textile wastewater (ITW) treatment plant showed that 1.5 ton of excess waste sludge, containing 304.5 Kg of dyes, are daily produced and discharged in landfills. Therefore, this by-product of activated sludge process (ASP) presents a serious environmental problem. In this work, a laboratory and pilot scale investigations were carried out to optimize aerobic biodegradation efficiency to reduce the amount of residual adsorbed dye that will be found in the waste sludge. The resistance of acclimated biomass to the toxicity of ITW was studied in 2.5 L batch reactors using different dye to biomass (D/B) ratios of 0.102, 0.25 and 0.72 g CODS/g VSS. Results of respirometric analyses showed that acclimated activated sludge (AS) biomass is able to treat ITW at high D/B ratio of 0.72 g CODS/g VSS. Moreover, biodegradation kinetic study using Monod law showed that COD and color removal were better for the highest D/B ratio. The half saturation coefficient of heterotrophs for indigo dye (KSind) of 20.01 g/m3 showed high affinity between biomass and dye molecules. Optimization of the process at pilot-scale with different hydraulic retention time (HRT) of 2-5 days, and different sludge recycling rates (SRR) of 220-680 m3/d, showed that high HRT of 5 days and a SRR of 0.22 allowed the best dye biodegradation efficiency (95%). Application of the best conditions at full-scale reduced significantly (89%) the amount of the discharged dyes from 304.5 Kg/d to 33 Kg/d. Results were numerically validated using a mathematical model based on the activated sludge model 1 (ASM1).

Fermentative hydrogen and methane co-production from anaerobic co-digestion of organic wastes at high loading rate coupling continuously and sequencing batch digesters.

The anaerobic co-digestion of the most abundant organic wastes was investigated for enhancing biogas production rate and quality. The used feedstock was composed of fruit and vegetable waste (FVW), waste-activated sludge (WAS), olive mill wastewater (OMW) and cattle manure (CM). A considerable methane yield of 340 L/kg volatile solid (VS) inlet was obtained using single-stage anaerobic sequencing batch reactors (ASBRs). However, VS biodegradation becomes difficult at high organic loading rate (OLR). Therefore, a continuously stirred tank reactor (CSTR) was integrated to the ASBR for waste pre-digestion. The dark fermentation leads to the improvement of organic matter solubilisation and bio-hydrogen productivity, reaching 0.73 L/L/day (H2 content of 49.8%) when pH decreased to 5.8. Therefore, methane productivity increased from 0.6 to 1.86 L/L/day in the methanogenic reactor with a better VS biodegradation (91.1%) at high OLR. Furthermore, the bio-hythane production was performed through a controlled biogas recirculation from the dark fermentation stage into the methaniser to reach 842.4 L/kg VS inlet. The produced biogas was composed of 8% H2, 28.5% CO2 and 63.5% CH4. Therefore, two-stage anaerobic co-digestion with coupled CH4 and H2 recuperation may be an important contribution for pollution control and high-rate bioenergy recovery (21.1 kJ/g VS inlet) from organic wastes.

Polyvinyl acetate processing wastewater treatment using combined Fenton's reagent and fungal consortium: Application of central composite design for conditions optimization.

The Fenton reaction as an oxidative degradation process was used for industrial chemical wastewater (ICW) pretreatment. The biodegradation of pretreated ICW was performed, in aqueous environment under aerobic condition, by a defined fungal consortium. The central composite design (CCD) was used to study the effect of nitrogen and phosphorus addition and the concentration of the pollution on the removal of polyvinyl alcohol (PVA) and organic compounds. The interaction between parameters was modeled using the response surface methodology (RSM). Results of optimization showed COD, PVA and color removal yields of 97.8%, 98.5% and 99.75%, respectively with a supplementof 1.4 gL-1 of (NH4)2SO4, 1.2 gL-1 of KH2PO4 and 75% of concentrated ICW. Enzymatic analysis proved that laccase and lignin peroxidase were involved in the biodegradation with 45 UIL-1 and 450 UIL-1, respectively. Furthermore, the analysis of metabolic products using Fourier transforms infrared spectroscopy (FTIR) and nuclear magnetic resonance (1HNMR) showed clearly the mineralization of organic compounds and the formation of formic acid and ethanol. Therefore, the effective treatment of ICW was achieved by developing an integrated chemical and biological process which met the requirement for a safety effluent respectful for environment without risks for public health.

Effect of enhancing nutrient balance in anaerobic digester feedstock by co-substrate addition on the microbial diversity and energy production from municipal sewage sludge.

Enhancement of methane production during anaerobic digestion of waste activated sludge (WAS) could improve the energy self sufficiency of the municipal wastewater treatment plants (WWTPs). Therefore, mixing WAS with organic wastes improved process performance and stability. In this work, the anaerobic co-digestion of WAS combined with the olive processing wastewater (OPW) was investigated and associated with the energetic benefits and microbial populations shifts. The bio-methane potential (BMP) of various WAS and OPW mixtures corresponding to increased phenols concentrations were tested. The anaerobic digestion of better proportions (90%/10% and 80%/20%) was performed in anaerobic sequencing batch reactors (ASBRs). The biodegradation of phenols at concentrations up to 0.76 g/L was confirmed by Sephadex gel filtration showing that ASBR, which is suspended growth reactor, can handle much higher concentration of toxic compounds. Microbial analysis showed that phenols induced significantly the archaea community dynamic, which showed highly richness and diversity in the well performed reactor. The dominant bacteria and archaea phylotypes were affiliated to Proteobacteria and Methanosarcinales, respectively. Therefore, OPW addition increased total energy production from 24.6 kWh/ton to 64.7 kWh/ton, which would provide 0.43 M€/year net benefits only from the electric power. In addition it brings a payback time on investment of 2 years for WWTPs modification, which was considered interesting.

Biohydrogen production from hyperthermophilic anaerobic digestion of fruit and vegetable wastes in seawater: Simplification of the culture medium of Thermotoga maritima.

Biohydrogen production by the hyperthermophilic and halophilic bacterium T. maritima, using fruit and vegetable wastes as the carbon and energy sources was studied. Batch fermentation cultures showed that the use of a culture medium containing natural seawater and fruit and vegetable wastes can replace certain components (CaCl2, MgCl2, Balch's oligo-elements, yeast extract, KH2PO4 and K2HPO4) present in basal medium. However, a source of nitrogen and sulfur remained necessary for biohydrogen production. When fruit and vegetable waste collected from a wholesale market landfill was used, no decreases in total H2 production (139 mmol L-1) or H2 yield (3.46 mol mol-1) was observed.

Improvement of activated sludge resistance to shock loading by fungal enzyme addition during textile wastewater treatment.

The effects of the additions of the fungal enzymatic extract were investigated in relation to the treatment of real textile wastewater (RTW) by the activated sludge process (ASP). The used enzyme cocktail was produced by a new isolated fungal Chaetomium globosum IMA1. The system that was operated with enzyme addition showed a better chemical oxygen demand (COD) removal efficiency (95%) compared to the control system (75%). In addition, the improvement of color removal (OD620) efficiencies was around 15%, when the newly consortium fungal enzymes was added. As the organic loading rate (OLR) increased from 0.33 g to 0.66 g COD L-1 d-1, a decrease in the performance of the two reactors was observed by monitoring the quality of treated effluents. However, the ASP working with enzyme addition showed a strong resistance to shock loadings and restored after few days compared to the control system, which was strongly inhibited. In fact, the enzyme addition improved the sludge volume index (SVI) and the activity of microorganisms. A high activity of laccase (300 U.L-1) enzyme was observed throughout the decolorization process in the improved system.

Effect of thermal pretreatment on the biogas production and microbial communities balance during anaerobic digestion of urban and industrial waste activated sludge.

The effect of thermal pre-treatment on the microbial populations balance and biogas production was studied during anaerobic digestion of waste activated sludge (WAS) coming from urban (US: urban sludge) and industrial (IS: industrial sludge) wastewater treatment plants (WWTP). The highest biogas yields of 0.42l/gvolatile solid (VS) removed and 0.37l/gVS removed were obtained with urban and industrial sludge pre-treated at 120°C, respectively. Fluorescent in situ hybridization (FISH) was used to quantify the major Bacteria and Archaea groups. Compared to control trails without pretreatment, Archaea content increased from 34% to 86% and from 46% to 83% for pretreated IS and US, respectively. In fact, the thermal pre-treatment of WAS enhanced the growth of hydrogen-using methanogens (HUMs), which consume rapidly the H2 generated to allow the acetogenesis. Therefore, the stable and better performance of digesters was observed involving the balance and syntrophic associations between the different microbial populations.

Industrial textile effluent decolourization in stirred and static batch cultures of a new fungal strain Chaetomium globosum IMA1 KJ472923.

The treatment of an industrial textile effluent (ITE) was investigated by using a mono-culture of a novel fungal strain Chaetomium globosum IMA1. This filamentous fungus was selected based on its capacity for dye removal via the biodegradation mechanism. The respirometric analysis showed that C. globosum IMA1 was resistant to an indigo concentration up to 700 mg equivalent COD/L. The decolourization of the ITE by C. globosum was performed in static and stirred batch systems. The better lignin peroxidase (LiP), laccase and the manganese peroxidase (MnP) productions were 829.9 U/L, 83 U/L and 247.8 U/L, respectively since 3-5 days under a stirred condition. Therefore, the chemical oxygen demand (COD) and colors (OD620) removal yields reached 88.4% and 99.8%, respectively. Fourier transforms infrared spectroscopy (FTIR) analysis of the treated effluent showed that the decolourization was due to the degradation and the transformation of dye molecules. However, spectrophotometric examination showed that the complete dye removal was through fungal adsorption (8%), followed by degradation (92%).

Microbial community structure associated with the high loading anaerobic codigestion of olive mill and abattoir wastewaters.

The effect of increasing the organic loading rates (OLRs) on the performance of the anaerobic codigestion of olive mill (OMW) and abattoir wastewaters (AW) was investigated under mesophilic and thermophilic conditions. The structure of the microbial community was also monitored. Increasing OLR to 9g of chemical oxygen demand (COD) L(-1)d(-1) affected significantly the biogas yield and microbial diversity at 35°C. However, at 55°C digester remained stable until OLR of 12g of CODL(-1)d(-1) with higher COD removal (80%) and biogas yield (0.52Lg(-1) COD removed). Significant differences in the bacterial communities were detected between mesophilic and thermophilic conditions. The dominant phyla detected in the digester at both phases were the Firmicutes, Actinobacteria, Bacteroidetes, Synergistetes and Spirochaete. However, Verrucomicrobia, Proteobacteria and the candidate division BRC1 were only detected at thermophilic conditions. The Methanobacteriales and the Thermoplasmales were found as a high predominant archaeal member in the anaerobic sludge.

Microbial ecology overview during anaerobic codigestion of dairy wastewater and cattle manure and use in agriculture of obtained bio-fertilisers.

The anaerobic co-digestion of dairy wastewater (DW) and cattle manure (CM) was examined and associated with microbial community's structures using Denaturing Gradient Gel Electrophoresis (DGGE). The highest volatile solids (VS) reduction yield of 88.6% and biogas production of 0.87 L/g VS removed were obtained for the C/N ratio of 24.7 at hydraulic retention time (HRT) of 20 days. The bacterial DGGE profile showed significant abundance of Uncultured Bacteroidetes, Firmicutes and Synergistetes bacterium. The Syntrophomonas strains were discovered in dependent association to H2-using bacteria such as Methanospirillum sp., Methanosphaera sp. and Methanobacterium formicicum. These syntrophic associations are essential in anaerobic digesters allow them to keep low hydrogen partial pressure. However, high concentrations of VFA produced from dairy wastes acidification allow the growth of Methanosarcina species. The application of the stabilised anaerobic effluent on the agriculture soil showed significant beneficial effects on the forage corn and tomato plants growth and crops.

Ecological significance of Synergistetes in the biological treatment of tuna cooking wastewater by an anaerobic sequencing batch reactor.

Lab-scale 2L-anaerobic sequencing batch reactor was operated under mesothermic conditions. The degradation of protein-rich organic matter was determined by chemical oxygen demand, biogas production, and protein-removal activity over the operation. The structure of the microbial community was determined by qPCR and next-generation sequencing on 16S rRNA genes. At the steady state, a very efficient removal of protein (92%) was observed. Our results demonstrate a decrease of archaeal and bacterial abundance over time. Members of the phylum Synergistetes, with a peculiar emphasis for those pertaining to families Dethiosulfovibrionaceae and Aminiphilaceae, are of major ecological significance regarding the treatment of this industrial wastewater. The prominent role to be played by members of the phylum Synergistetes regarding protein and/or amino acid degradation is discussed.

Aminobacterium thunnarium sp. nov., a mesophilic, amino acid-degrading bacterium isolated from an anaerobic sludge digester, pertaining to the phylum Synergistetes.

A new Gram-staining-positive, non-sporulating, mesophilic, amino acid-degrading anaerobic bacterium, designated strain OTA 102(T), was isolated from an anaerobic sequencing batch reactor treating wastewater from cooking tuna. The cells were curved rods (0.6-2.5×0.5 µm) and occurred singly or in pairs. The strain was motile by means of one lateral flagellum. Strain OTA 102(T) grew at temperatures between 30 and 45 °C (optimum 40 °C), between pH 6.0 and 8.4 (optimum pH 7.2) and NaCl concentrations between 1 and 5 % (optimum 2 %, w/v). Strain OTA 102(T) required yeast extract for growth. Serine, threonine, glycine, cysteine, citrate, fumarate, α-ketoglutarate and pyruvate were fermented. When co-cultured with Methanobacterium formicicum as the hydrogen scavenger, strain OTA 102(T) oxidized alanine, valine, leucine, isoleucine, aspartate, tyrosine, methionine, histidine and asparagine. The genomic DNA G+C content of strain OTA 102(T) was 41.7 mol%. The main fatty acid was iso-C15 : 0. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain OTA 102(T) was related to Aminobacterium colombiense and Aminobacterium mobile (95.5 and 95.2 % similarity, respectively), of the phylum Synergistetes. On the basis of phylogenetic, genetic and physiological characteristics, strain OTA 102(T) is proposed to represent a novel species of the genus Aminobacterium, Aminobacterium thunnarium sp. nov. The type strain is OTA 102(T) ( = DSM 27500(T) = JCM 19320(T)).

Performance study of biofilter developed to treat H2S from wastewater odour.

Biofiltration is an efficient biotechnological process used for waste gas abatement in various industrial processes. It offers low operating and capital costs and produces minimal secondary waste streams. The objective of this study was to evaluate the performance of a pilot scale biofilter in terms of pollutants' removal efficiencies and the bacterial dynamics under different inlet concentrations of H2S. The treatment of odourous pollutants by biofiltration was investigated at a municipal wastewater treatment plant (WWTP) (Charguia, Tunis, Tunisia). Sampling and analyses were conducted for 150 days. Inlet H2S concentration recorded was between 200 and 1300 mg H2S.m(-3). Removal efficiencies reached 99% for the majority of the running time at an empty bed retention time (EBRT) of 60 s. Heterotrophic bacteria were found to be the dominant microorganisms in the biofilter. The bacteria were identified as the members of the genus Bacillus, Pseudomonas and xanthomonadacea bacterium. The polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) method showed that bacterial community profiles changed with the H2S inlet concentration. Our results indicated that the biofilter system, containing peat as the packing material, was proved able to remove H2S from the WWTP odourous pollutants.

Isolation and characterization of Desulfocurvus thunnarius sp. nov., a sulfate-reducing bacterium isolated from an anaerobic sequencing batch reactor treating cooking wastewater.

A novel anaerobic, chemo-organotrophic, sulfate-reducing bacterium, designated strain Olac 40(T), was isolated from a Tunisian wastewater digestor. Cells were curved, motile rods or vibrios (5.0-7.0×0.5 µm). Strain Olac 40(T) grew at temperatures between 15 and 50 °C (optimum 40 °C), and between pH 5.0 and 9.0 (optimum pH 7.1). It did not require NaCl for growth but tolerated it up to 50 g l(-1) (optimum 2 g l(-1)). In the presence of sulfate or thiosulfate, strain Olac 40(T) used lactate, pyruvate and formate as energy sources. Growth was observed on H2 only in the presence of acetate as carbon source. In the presence of sulfate or thiosulfate, the end products of lactate oxidation were acetate, sulfide and CO2. Sulfate, thiosulfate and sulfite were used as terminal electron acceptors, but not elemental sulfur, nitrate or nitrite. The genomic DNA G+C content of strain Olac 40(T) was 70 mol%. The profile of polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and four phospholipids. The main fatty acids were C16 : 0, anteiso-C15 : 0 and iso-C15 : 0. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Olac 40(T) was affiliated with the family Desulfovibrionaceae within the class Deltaproteobacteria. On the basis of 16S rRNA gene sequence comparisons and physiological characteristics, strain Olac 40(T) is proposed to be assigned to a novel species of the genus Desulfocurvus, for which the name Desulfocurvus thunnarius is proposed. The type strain is Olac 40(T) ( = DSM 26129(T) = JCM 18546(T)).

An improved strategy for easy process monitoring and advanced purification of recombinant proteins.

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.

Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters.

The performance of anaerobic digestion of abattoir wastewaters (AW) in an upflow anaerobic filter (UAF) was investigated under mesophilic (37°C) and thermophilic (55°C) conditions. The effects of increasing temperature on the performance of the UAF and on the dynamics of the microbial community of the anaerobic sludge were studied. The results showed that chemical oxygen demand (COD) removal efficiency of 90% was achieved for organic loading rates (OLRs) up to 4.5g CODL(-1)d(-1) in mesophilic conditions, while in thermophilic conditions, the highest OLRs of 9 g CODL(-1)d(-1) led to the efficiency of 72%. The use of molecular and microbiological methods to recover microbial populations involved in this process showed that fermentative bacteria were the prominent members of the sludge microbial community. Three novel strains were identified as Macellibacteroides fermentans, Desulfotomaculum peckii and Defluviitalea saccharophila.

Biological sludge reduction during abattoir wastewater treatment process using a sequencing batch aerobic system.

Excess sludge disposal during biological treatment of wastewater is subject to numerous constraints, including social, health and regulatory factors. To reduce the amount of excess sludge, coupled processes involving different biological technologies are currently under taken. This work presents a laboratory scale sequencing batch aerobic system included an anaerobic zone for biomass synchronization (SBAAS: sequencing batch aerobic anaerobic system). This system was adopted to reduce sludge production during abattoir wastewater (AW) treatment. The average chemical oxygen demand (COD) removal efficiency of 89% was obtained at a hydraulic retention time (HRT) and a sludge retention time (SRT) of 2 days and 15-20 days, respectively. The comparison of SBAAS performances with a conventional sequencing batch activated sludge system (SBASS) found that the observed biomass production yield (Y(obs)) were in the ranges of 0.26 and 0.7 g suspended solids g(-1) COD removed, respectively. A significant reduction in the excess biomass production of 63% was observed by using the SBAAS. In fact, in the anaerobic zone microorganisms consume the intracellular stocks of energy by endogenous metabolism, which limits biosynthesis and accelerates sludge decay. The single strand conformation polymorphism (SSCP) method was used to study the dynamic and the diversity of bacterial communities. Results showed a significant change in the population structure by including the anaerobic stage in the process, and revealed clearly that the sludge production yield can be correlated with the bacterial communities present in the system.

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins.

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.

Performance of a submerged membrane bioreactor for the aerobic treatment of abattoir wastewater.

The performance of a submerged membrane bioreactor (SMBR) has been investigated for abattoir wastewater (AW) treatment. The chemical oxygen demand (COD) of permeate has not exceeded 25 mg L(-1) providing an average COD removal of 98%. Microbiological analysis showed that the SMBR has allowed a complete removal of fecal coliforms, Listeria and Salmonella. A significant reduction in the excess biomass production was also observed. In fact, the yield of biomass production (Yobs) ranged between 0 and 0.106 g suspended solids/g COD removed. The study of the dynamic of bacterial communities using the single strand conformation polymorphism (SSCP) method showed a significant change in the population structure and revealed a correlation between the sludge production yield and the bacterial communities.