Improving the storage of cover crops by co-ensiling with different waste types: Effect on fermentation and effluent production.

Cover crops harvested at a low maturity stage generally have a high moisture content, which may generate energy losses during silage storage via effluent production and undesirable fermentations. This paper investigates the use of different waste types as absorbent co-substrates to be added before ensiling. The relation between the absorbent water holding capacity and silage effluent volume was first studied to find an effective parameter to prevent effluent production. Effluent retention was found to be proportional to the absorbent loading and water holding capacity (r2 = 0.98) and up to 90 % of effluent production was avoided when compared to control (295 l.t-1). The impact of different co-substrates (including bio-waste and manures) on overall ensiling performances was then investigated at an optimized absorbent loading. All co-substrates allowed a total effluent retention while a 76 l.t-1 effluent volume was reported for the control. The silage fermentation was modified or mostly unchanged depending on the co-substrate chemical and microbial properties and different metabolic pathways were observed (e.g. homolactic or butyric fermentation). In most conditions, the methane potential of the crop was efficiently preserved over a storage of 60 days. Co-ensiling was shown to be a relevant silage preparation method for biogas production.

Conditions for efficient alkaline storage of cover crops for biomethane production.

An innovative process aiming to combine storage and alkali pretreatment of cover crops was investigated using lime as a low cost and environmental friendly reactant. Different lime loadings and Total Solid concentrations (TS) allowed to highlight the abiotic mechanisms of deacetylation during the early stages of the process. Long-term storage experiments of rye and sunflower cover crops at 100 g.kgTS-1 lime loading allowed to evaluate the fermentation kinetics and to compare performances in dry and wet conditions to classical silage storage. The dry condition allowed an efficient alkaline storage and up to a 15.7% Biochemical Methane Potential (BMP) increase, while the wet condition underwent a succession of fermentations with a high butyric acid accumulation and H2 production, leading to a 13% BMP loss. Silage experiments allowed an efficient preservation of the BMP, with no significant variation over the 6-month storage duration.

Microbial community redundance in biomethanation systems lead to faster recovery of methane production rates after starvation.

The Power-to-Gas concept corresponds to the use of the electric energy surplus to produce H2 by water electrolysis, that can be further converted to methane by biomethanation. However, the fluctuant production of renewable energy sources can lead to discontinuous H2 injections into the reactors, that may interfere with the adaptation of the microbial community to high H2 partial pressures. In this study, the response of the microbial community to H2 and organic feed starvation was evaluated in in-situ and ex-situ biomethanation. The fed-batch reactors were fed with acetate or glucose and H2, and one or four weeks of starvation periods were investigated. Methane productivity was mostly affected by the four-week starvation period. However, both in-situ and ex-situ biomethanation reactors recovered their methane production rate after starvation within approximately one-week of normal operation, while the anaerobic digestion (AD) reactors did not recover their performances even after 3 weeks of normal operation. The recovery failure of the AD reactors was probably related to a slow growth of the syntrophic and methanogen microorganisms, that led to a VFA accumulation. On the contrary, the faster recovery of both biomethanation reactors was related to the replacement of Methanoculleus sp. by Methanobacterium sp., restoring the methane production in the in-situ and ex-situ biomethanation reactors. This study has shown that biomethanation processes can respond favourably to the intermittent H2 addition without compromising their CH4 production performance.

Long term alkaline storage and pretreatment process of cover crops for anaerobic digestion.

The aim of this work was to study an innovative alkaline process on two cover crops. CaO load of 60 g.kgTS-1 was implemented to combine the functions of storage and pretreatment. Lab-scale reactors were monitored for 180 days to assess the effect of this process on the physico-chemical properties of the biomass. From the first days, pH was not maintained in an alkaline zone and microbial fermentation activity was observed with the degradation of available carbohydrates and production of metabolites, CO2 and H2. High butyric acid accumulation was observed and mass losses of 18.1% and 9.0% of initial VS occurred for oat and rye, respectively. However, no methane potential loss was recorded in the short and long term and the crops were efficiently preserved. The pretreatment had no major impact on fiber solubilization, and no increase in BMP was obtained, which was attributed to the short duration of the alkaline conditions.

Role of indigenous bacteria in dark fermentation of organic substrates.

Hydrogen production by dark fermentation of complex organic substrates, such as biowaste, can naturally take place with indigenous bacteria or by adding an external microbial inoculum issued from various natural environments. This study aims to determine whether indigenous bacteria associated with thermal pretreatment could impact dark fermentation performances. Biochemical hydrogen potential tests were carried out on seven organic substrates. Results showed a strong influence of the indigenous bacteria which are as effective as thermally pretreated exogenous bacteria to produce H2 and metabolites. High abundance in Clostridiales and/or Enterobacteriales was associated with high H2 yield. This study shows that no inoculum nor pretreatment are required to achieve satisfactory dark fermentation performances from organic waste.

Process type is the key driver of the fate of organic micropollutants during industrial scale treatment of organic wastes.

Organic micropollutants (OMPs) such as polycyclic aromatic hydrocarbons, nonylphenols and pharmaceutical products are ubiquitous in organic wastes generated by most human activities. Those wastes are mainly recycled by land spreading, most often after treatments, such as liming, dewatering, composting or anaerobic digestion. It has been shown essentially at lab scales that biological treatments have an effect on the removal of some OMPs. However, less is known on the role of each step of industrial treatment lines combining physico-chemical and biological treatments on the OMP fate and removal. The present study focuses on the impact of waste treatment on the fate of 53 OMPs along 10 industrial treatment lines treating urban, agricultural wastes or mixtures. The combination of studying a diversity of organic wastes and of OMPs with different characteristics (solubility, ionic charges, hydrophobicity etc.), sampling in situ industrial sites, quantifying native OMP concentrations and looking at each step of complete treatment lines allows for a global and representative view of the OMP fate in the French organic waste treatment sector. Less studied wastes, i.e. territorial mixtures, revealed intermediate OMP contents and compositions, between urban and agricultural wastes. Dewatering and liming, usually dismissed, had a noticeable effect on concentrations. Anaerobic digestion and composting had significant effects on the removal of all pollutant families. Combination of processes enhanced most OMP dissipation. Here we showed for the first time that the process type rather than the waste origin affects dissipation of organic micropollutants. Such data could be used to build and validate dynamic models for the fate of OMPs on solid waste treatment plants.

Optimal conditions for flexible methane production in a demand-based operation of biogas plants.

The aim of the presented work was to study the methane production limits and to determine optimal conditions for flexible operation of an anaerobic reactor in order to set up an operational strategy. Punctual overloads were conducted in a laboratory-scale anaerobic reactor with readily biodegradable solid substrates, and the influences of overload intensity, baseload value and substrate used were investigated. A maximal daily value around 1000mL/L of reactor for methane production has been assessed. This value did not evolve significantly during experiment time, and conditioned the persistence of overloads as well as the flexibility margin on the reactor, which ranged from +25% to +140% on daily production. Results highlighted the fact that for a maximum flexibility, low organic loading rates are better to work with on this type of reactors.

Electrical conductivity as a state indicator for the start-up period of anaerobic fixed-bed reactors.

The aim of this work was to analyse the applicability of electrical conductivity sensors for on-line monitoring the start-up period of an anaerobic fixed-bed reactor. The evolution of bicarbonate concentration and methane production rate was analysed. Strong linear relationships between electrical conductivity and both bicarbonate concentration and methane production rate were observed. On-line estimations of the studied parameters were carried out in a new start-up period by applying simple linear regression models, which resulted in a good concordance between both observed and predicted values. Electrical conductivity sensors were therefore identified as an interesting method for monitoring the start-up period of anaerobic fixed-bed reactors due to its reliability, robustness, easy operation, low cost, and minimum maintenance compared with the currently used sensors.

Bioelectrochemical treatment of table olive brine processing wastewater for biogas production and phenolic compounds removal.

Industry of table olives is widely distributed over the Mediterranean countries and generates large volumes of processing wastewaters (TOPWs). TOPWs contain high levels of organic matter, salt, and phenolic compounds that are recalcitrant to microbial degradation. This work aims to evaluate the potential of bioelectrochemical systems to simultaneously treat real TOPWs and recover energy. The experiments were performed in potentiostatically-controlled single-chamber systems fed with real TOPW and using a moderate halophilic consortium as biocatalyst. In conventional anaerobic digestion (AD) treatment, ie. where no potential was applied, no CH4 was produced. In comparison, Bio-Electrochemical Systems (BES) showed a maximum CH4 yield of 701 ± 13 NmL CH4·LTOPW(-1) under a current density of 7.1 ± 0.4 A m(-2) and with a coulombic efficiency of 30%. Interestingly, up to 80% of the phenolic compounds found in the raw TOPW (i.e. hydroxytyrosol and tyrosol) were removed. A new theoretical degradation pathway was proposed after identification of the metabolic by-products. Consistently, microbial community analysis at the anode revealed a clear and specific enrichment in anode-respiring bacteria (ARB) from the genera Desulfuromonas and Geoalkalibacter, supporting the key role of these electroactive microorganisms. As a conclusion, bioelectrochemical systems represent a promising bioprocess alternative for the treatment and energy recovery of recalcitrant TOPWs.

Conservation of acquired morphology and community structure in aged biofilms after facing environmental stress.

The influence of growth history on biofilm morphology and microbial community structure is poorly studied despite its important role for biofilm development. Here, biofilms were exposed to a change in hydrodynamic conditions at different growth stages and we observed how biofilm age affected the change in morphology and bacterial community structure. Biofilms were developed in two bubble column reactors, one operated under constant shear stress and one under variable shear stress. Biofilms were transferred from one reactor to the other at different stages in their development by withdrawing and inserting the support medium from one reactor to the other. The developments of morphology and microbial community structure were followed by image analysis and molecular tools. When transferred early in biofilm development, biofilms adapted to the new hydrodynamic conditions and adopted features of the biofilm already developed in the receiving reactor. Biofilms transferred at a late state of biofilm development continued their initial trajectories of morphology and community development even in a new environment. These biofilms did not immediately adapt to their new environment and kept features acquired during their early growth phase, a property we called memory effect.

Dynamic observation of the biodegradation of lignocellulosic tissue under solid-state anaerobic conditions.

The solid-state anaerobic digestion (SS-AD) of wheat straw was characterized under low inoculated batch tests during 244 days. High levels of degradation of the cellulose (52%±1) and hemicelluloses (55%±2) were observed at the final stages and associated to a methane yield of 204±16 NmL gTS(-1). Ultrastructural observations, using transmission electronic microscopy, indicated that microorganisms degraded wheat straw from the central to the outer tissue (i.e. parenchyma to epidermis), depending on cell chemical, physical accessibility and the degree of lignification. Furthermore, major degradation of sclerenchyma secondary walls was observed. The bioaccessibility of lignocellulosic structures of wheat straw is mainly limited by the external waxy layer (cuticle), tertiary cell walls, high silica content and access to the cell lumen.

Substrate milling pretreatment as a key parameter for Solid-State Anaerobic Digestion optimization.

The effect of milling pretreatment on performances of Solid-State Anaerobic Digestion (SS-AD) of raw lignocellulosic residue is still controverted. Three batch reactors treating different straw particle sizes (milled 0.25 mm, 1 mm and 10 mm) were followed during 62 days (6 sampling dates). Although a fine milling improves substrate accessibility and conversion rate (up to 30% compared to coarse milling), it also increases the risk of media acidification because of rapid and high acids production during fermentation of the substrate soluble fraction. Meanwhile, a gradual adaptation of microbial communities, were observed according to both reaction progress and methanogenic performances. The study concluded that particle size reduction affected strongly the performances of the reaction due to an increase of substrate bioaccessibility. An optimization of SS-AD processes thanks to particle size reduction could therefore be applied at farm or industrial scale only if a specific management of the soluble compounds is established.

An automated method for the quantification of moving predators such as rotifers in biofilms by image analysis.

In natural environments as well as in industrial processes, microorganisms form biofilms. Eukaryotic microorganisms, like metazoans and protozoans, can shape the microbial communities because of their grazing activity. However, their influence on biofilm structure is often neglected because of the lack of appropriate methods to quantify their presence. In the present work, a method has been developed to quantify moving population of rotifers within a biofilm. We developed an automated approach to characterize the rotifer population density. Two time lapse images are recorded per biofilm location at an interval of 1s. By subtracting the two images from each other, rotifer displacements that occurred between the two images acquisition can be quantified. A comparison of the image analysis approach with manually counted rotifers showed a correlation of R(2)=0.90, validating the automated method. We verified our method with two biofilms of different superficial and community structures and measured rotifer densities of up to 1700 per cm(2). The method can be adapted for other types of moving organisms in biofilms like nematodes and ciliates.

New urban wastewater treatment with autotrophic membrane bioreactor at low chemical oxygen demand/N substrate ratio.

The potential for total nitrogen removal from municipal wastewater has been evaluated in an autotrophic membrane bioreactor running with a low chemical oxygen demand (COD)/N ratio to simulate its combination with an upstream physicochemical process that retains a large proportion of organic matter. The tests were conducted in a laboratory scale submerged membrane bioreactor loaded with a synthetic influent. Nitrogen loading rate was 0.16 kgN-NH4+.m(-3).d(-1) and sodium acetate was added as a carbon source. Results have shown that nitrogen elimination can reach 85% for a COD/N ratio of 5, with COD removal exceeding 97%. However, a COD/N ratio of 3.5 was found to be the limiting factor for successfully reaching the overall target value of 10 mgN.L(-1) in the effluent. Nevertheless, low COD/N ratios make it possible to work with low total suspended solid concentrations in the bioreactor, which greatly facilitates membrane fouling control by a simple aeration and backwashing strategy.

Modelling ammonium-oxidizing population shifts in a biofilm reactor.

The dynamic reactor behaviour of a nitrifying inverse turbulent bed reactor, operated at varying loading rate, was described with a one-dimensional two-step nitrification biofilm model. In contrast with conventional biofilm models, this model includes the competition between two genetically different populations of ammonia-oxidizing bacteria (AOB), besides nitrite-oxidizing bacteria (NOB). Previously gathered experimental evidence showed that different loading rates in the reactor resulted in a change in the composition of the AOB community, besides a different nitrifying performance. The dissolved oxygen concentration in the bulk liquid was put forward as the key variable governing the experimentally observed shift from Nitrosomonas europaea (AOB1) to Nitrosomonas sp. (AOB2), which was confirmed by the developed one-dimensional biofilm model. Both steady state and dynamic analysis showed that the influence of microbial growth and endogenous respiration parameters as well as external mass transfer limitation have a clear effect on the competition dynamics. Overall, it was shown that the biomass distribution profiles of the coexisting AOB reflected the ecological niches created by substrate gradients.

Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion.

Among all the process parameters of solid-state anaerobic digestion (SS-AD), total solid content (TS), inoculation (S/X ratio) and size of the organic solid particles can be optimized to improve methane yield and process stability. To evaluate the effects of each parameter and their interactions on methane production, a three level Box-Behnken experimental design was implemented in SS-AD batch tests degrading wheat straw by adjusting: TS content from 15% to 25%, S/X ratio (in volatile solids) between 28 and 47 and particle size with a mean diameter ranging from 0.1 to 1.4mm. A dynamic analysis of the methane production indicates that the S/X ratio has only an effect during the start-up phase of the SS-AD. During the growing phase, TS content becomes the main parameter governing the methane production and its strong interaction with the particle size suggests the important role of water compartmentation on SS-AD.

Distribution and hydrophobic properties of Extracellular Polymeric Substances in biofilms in relation towards cohesion.

A heterotrophic biofilm (B1) and a mixed autotrophic-heterotrophic biofilm (B2) were developed in an annular reactor and submitted to an erosion test in order to selectively detach top layers from the bottom layers. Densities of the basal layers were 5-fold higher and 3-fold higher than the densities of the entire biofilms B1 and B2, respectively. After extraction, EPS content in B1 biofilm was found higher in the basal layer (95 mg g⁻¹ VSS) compared to the top layer (30 mg g⁻¹ VSS), while B2 biofilm had a higher EPS content in the top layer (303 mg g⁻¹ VSS) compared to the basal layer (135 mg g⁻¹ VSS). Hydrophobic Interaction Chromatography (HIC) indicates that hydrophobic EPS (HEPS) in both biofilms reached 21% of EPS in basal cohesive layers, and remained slightly lower or identical (16-19%) in top detached biofilm layers. Strong interacting HEPS were found in a higher proportion in the mixed autotrophic-heterotrophic B2 which was also more diversified in terms of bacterial populations than the B1 heterotrophic biofilm. These results show that HEPS content correlates better with cohesive properties of the biofilm layers than global EPS content and that strong hydrophobic adhesion forces may be related to microbial populations such as the presence of nitrifiers.

Biofilm model calibration and microbial diversity study using Monte Carlo simulations.

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single-species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single-species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single-species biofilm model was then extended to a multi-species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/KS ). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution.

Effect of organic loading rate on anaerobic digestion of thermally pretreated Scenedesmus sp. biomass.

Biogas production is one of the means to produce a biofuel from microalgae. Biomass consisting mainly of Scenedesmus sp. was thermally pretreated and optimum pretreatment length (1 h) and temperature (90 °C) was selected. Different chemical composition among batches stored at 4 °C for different lengths of time resulted in organic matter hydrolysis percentages ranging from 3% to 7%. The lower percentages were attributed to cell wall thickening observed during storage for 45 days. The different hydrolysis percentages did not cause differences in anaerobic digestion. Pretreatment of Scenedesmus sp. at 90 °C for 1h increased methane production 2.9 and 3.4-fold at organic loading rates (OLR) of 1 and 2.5 kg COD m(-3) day(-1), respectively. Regardless the OLR, inhibition caused by organic overloading or ammonia toxicity were not detected. Despite enhanced methane production, anaerobic biodegradability of this biomass remained low (32%). Therefore, this microalga is not a suitable feedstock for biogas production unless a more suitable pretreatment can be found.

Heterogeneity and spatial distribution of bacterial background contamination in pulp and process water of a paper mill.

Identifying the source and the distribution of bacterial contaminant communities in water circuits of industrial applications is critical even when the process may not show signs of acute biofouling. The endemic contamination of facilities can cause adverse effects on process runability but may be masked by the observed daily variability. The distribution of background communities of bacterial contaminants may therefore be critical in the development of new site-specific antifouling strategies. In a paper mill as one example for a full-scale production process, bacterial contaminants in process water and pulp suspensions were mapped using molecular fingerprints at representative locations throughout the plant. These ecological data were analyzed in the process-engineering context of pulp and water flow in the facilities. Dispersal limits within the plant environment led to the presence of distinct groups of contaminant communities in the primary units of the plant, despite high flows of water and paper pulp between units. In the paper machine circuit, community profiles were more homogeneous than in the other primary units. The variability between sampled communities in each primary unit was used to identify a possible point source of microbial contamination, in this case a storage silo for reused pulp. Part of the contamination problem in the paper mill is likely related to indirect effects of microbial activity under the local conditions in the silo rather than to the direct presence of accumulated microbial biomass.