Medium chain Fatty acids production from Food Waste via homolactic fermentation and lactate/ethanol elongation: Electron balance and thermodynamic assessment.

This study explored the potential of Food Waste (FW) extract as a suitable substrate for Medium Chain Fatty Acids (MCFAs) production, in a single-phase reactor, where both fermentation and Chain Elongation (CE) processes occurred simultaneously. A continuous experiment was conducted with an Organic Loading Rate (OLR) = 20 gCOD L-1 d-1 and was fed in batch mode twice a week with pH = 6. In addition, four batch tests were performed, to assess the effects on the MCFAs production of caproate inhibition, hydrogen partial pressure (PH2) and different lactate/acetate ratios. Thermodynamics and electron flux were calculated to gain insights into the process pathways. Due to the presence of aminoacids, fermentation was mostly homolactic and both lactate and ethanol were produced as Electron Donors (EDs); the average MCFAs production efficiency was âˆ¼ 12 %, although after 4 weeks the elongation process was halted, resulting in EDs accumulation. This occurred regardless of inoculum selection and the presence of caproate as a possible inhibitor, suggesting that EDs accumulation was due to the elongation process kinetics being slower than those of the fermentation step, thus calling for a longer Hydraulic Retention Time (HRT). It's worth noting that lactate was prevalently self-elongated to butyrate, whereas ethanol elongation only took place after lactate depletion, but was more efficient since it required other Electron Acceptors (EAs) such as butyrate, propionate or valerate. Moreover, the selected pH limited the acrylate pathway to a reasonable extent, whereas the high PH2 prevented both ethanol and lactate oxydation to acetate.

Thermally enhanced solid-liquid separation process in food waste biorefinery: modelling the anaerobic digestion of solid residues.

The biochemical valorization potential of food waste (FW) could be exploited by extracting decreasing added-value bio-based products and converting the final residues into energy. In this context, multi-purpose and versatile schemes integrating thermal and biochemical conversion processes will play a key role. An upstream thermal pretreatment + solid-liquid separation unit was here proposed to optimize the conversion of the liquid fraction of FW into valuable chemicals through semi-continuous fermentation process, and the conversion of the residual solid fraction into biomethane through anaerobic digestion. The solid residues obtained after thermal pretreatment presented a higher soluble COD fraction, which resulted in higher methane production with respect to the raw residues (0.33 vs. 0.29 Nm3CH4 kg-1VSfed) and higher risk of acidification and failure of methanogenesis when operating at lower HRT (20d). On the contrary, at HRT = 40 d, the pretreatment did not affect the methane conversion rates and both tests evidenced similar methane productions of 0.33 Nm3CH4 kg-1VSfed. In the reactor fed with pretreated residue, the association of hydrogenotrophic methanogens with syntrophic bacteria prevented the acidification of the system. Modelling proved the eligibility of the FW solid residues as substrates for anaerobic digestion, given their small inert fractions that ranged between 0% and 30% of the total COD content.

Ecology of food waste chain-elongating microbiome.

Microbial chain elongation has emerged as a valuable bioprocess for obtaining marketable products, such as medium chain fatty acids usable in several industrial applications, from organic waste. The understanding of the microbiology and microbial ecology in these systems is crucial to apply these microbiomes in reliable production processes controlling microbial pathways to promote favourable metabolic processes, which will in turn increase product specificity and yields. In this research, the dynamics, cooperation/competition and potentialities of bacterial communities involved in the long-term lactate-based chain elongation process from food waste extract were evaluated under different operating conditions by DNA/RNA amplicon sequencing and functional profile prediction. The feeding strategies and the applied organic loading rates strongly affected the microbial community composition. The use of food waste extract promoted the selection of primary fermenters (i.e., Olsenella, Lactobacillus) responsible for the in situ production of electron donors (i.e., lactate). The discontinuous feeding and the organic loading rate 15 gCOD L-1 d-1 selected the best performing microbiome in which microbes coexist and cooperate to complete the chain elongation process. Both at DNA and RNA level, this microbiome was composed by the lactate producer Olsenella, the short chain fatty acids producers Anaerostipes, Clostridium sensu stricto 7, C. sensu stricto 12, Corynebacterium, Erysipelotrichaceae UCG-004, F0332, Leuconostoc, and the chain elongator Caproiciproducens. This microbiome also showed the highest predicted abundance of short-chain acyl-CoA dehydrogenase, the functional enzyme responsible for the chain elongation process. The combined approach herein used allowed to study the microbial ecology of chain elongation process from food waste by identifying the main functional groups, establishing the presence of potential biotic interactions within the microbiomes, and predicting metabolic potentialities. This study provided pivotal indications for the selection of high-performance microbiome involved in caproate production from food waste that can serve as a basis for further improving system performance and engineering the process scale-up.

Biorefining food waste through the anaerobic conversion of endogenous lactate into caproate: A fragile balance between microbial substrate utilization and product inhibition.

New technologies development and renewable source exploitation are key tools to realize the European Green Deal and to boost the bio-based economy. In this context, fermentation of organic residues as food waste is an efficient method to obtain marketable products such as carboxylic acids widely applied in industrial production. Under favourable thermodynamic conditions, short chain fatty acids deriving from primary fermentation could be biologically converted into medium-chain fatty acids as caproate via chain elongation (CE) process, by using ethanol or lactate as electron donors. This study evaluates the effectivity of producing caproate from Food Waste extract rich in organics with in situ electron donor production. The test carried out at OLR 15 gCOD L-1d-1 showed high Volatile Fatty Acids (from acetic to caproic acid) yields (0.37 g g-1CODfed), with a maximum caproate concentration of 8 g L-1. The associated microbiome was composed by lactate-producing bacteria (Corynebacterium, Lactobacillus, and Olsenella) and by chain elongators (Clostridiaceae and Caproiciproducens). By stressing the system with OLR increase up to 20 gCOD L-1d-1, the CE process was inhibited by the high concentration of caproate (low occurrence of Clostridiaceae and Caproiciproducens). Nevertheless, after few days of stop-feeding regime imposed to the system, the microbiome restored its capability to proceed with lactate-based CE pathways. Different batch tests carried out with the inhibited biomass at increasing initial caproate concentration confirmed its impact on lactate utilization kinetics.

Insights into the Anaerobic Hydrolysis Process for Extracting Embedded EPS and Metals from Activated Sludge.

The amount of sewage sludge generated from wastewater treatment plants globally is unavoidably increasing. In recent years, significant attention has been paid to the biorefinery concept based on the conversion of waste streams to high-value products, material, and energy by microorganisms. However, one of the most significant challenges in the field is the possibility of controlling the microorganisms' pathways in the anaerobic environment. This study investigated two different anaerobic fermentation tests carried out with real waste activated sludge at high organic loading rate (10 g COD L-1d-1) and short hydraulic retention time (HRT) to comprehensively understand whether this configuration enhances extracellular polymeric substance (EPS) and metal solubilisation. The quantity of EPS recovered increased over time, while the chemical oxygen demand to EPS ratio remained in the range 1.31-1.45. Slightly acidic conditions and sludge floc disintegration promoted EPS matrix disruption and release, combined with the solubilisation of organically bound toxic metals, such as As, Be, Cu, Ni, V, and Zn, thereby increasing the overall metal removal efficiency due to the action of hydrolytic microorganisms. Bacteroidetes, Firmicutes, and Chloroflexi were the most abundant phyla observed, indicating that the short HRT imposed on the systems favoured the hydrolytic and acidogenic activity of these taxa.

Microbial Community Successional Changes in a Full-Scale Mesophilic Anaerobic Digester from the Start-Up to the Steady-State Conditions.

Anaerobic digestion is a widely used technology for sewage sludge stabilization and biogas production. Although the structure and composition of the microbial communities responsible for the process in full-scale anaerobic digesters have been investigated, little is known about the microbial successional dynamics during the start-up phase and the response to variations occurring in such systems under real operating conditions. In this study, bacterial and archaeal population dynamics of a full-scale mesophilic digester treating activated sludge were investigated for the first time from the start-up, performed without adding external inoculum, to steady-state operation. High-throughput 16S rRNA gene sequencing was used to describe the microbiome evolution. The large majority of the reads were affiliated to fermentative bacteria. Bacteroidetes increased over time, reaching 22% of the total sequences. Furthermore, Methanosaeta represented the most abundant methanogenic component. The specific quantitative data generated by real-time PCR indicated an enrichment of bacteria and methanogens once the steady state was reached. The analysis allowed evaluation of the microbial components more susceptible to the shift from aerobic to anaerobic conditions and estimation of the microbial components growing or declining in the system. Additionally, activated sludge was investigated to evaluate the microbial core selected by the WWTP operative conditions.

A novel cascade biorefinery approach to transform food waste into valuable chemicals and biogas through thermal pretreatment integration.

A novel biorefinery platform integrating thermal pretreatment and solid-liquid separation unit is here proposed to fully exploit food waste (FW) potential for production of valuable chemicals and energy through semi-continuous anaerobic bioconversion. The liquid fraction deriving from raw or pretreated FW, was fermented into volatile fatty acids (VFAs, from acetic to caproic acid) while the residual fraction was converted into biomethane. Thermal pretreatment effectively extracted a portion of the macromolecular organics, especially starch, to the liquid phase, promoting acidogenic fermentation and chain elongation pathways (0.43 gVFA g-1VSfed and 0.58 gVFA g-1VSfed with raw and pretreated extract, respectively). In parallel, anaerobic digestion of solid residue in 10 L reactors showed process stability and higher conversion rate for the pretreated residue (0.31 against 0.26 Nm3CH4 kg-1VSfed). The mass-transfer balance coupled with the economic assessment, calculated in terms of direct gross added value, indicated promising revenues by integrating the thermal upstream treatment.

Direct Conversion of Food Waste Extract into Caproate: Metagenomics Assessment of Chain Elongation Process.

In a circular economy strategy, waste resources can be used for the biological production of high added-value substances, such as medium chain fatty acids (MCFAs), thus minimising waste and favouring a sustainable process. This study investigates single-stage fermentation processes for the production of MCFAs in a semi-continuous reactor treating the extract of real food waste (FW), without the addition of external electron donors. Two sequential acidogenic fermentation tests were carried out at an organic loading rate (OLR) of 5 and 15 gCOD L-1d-1 with a hydraulic retention time of 4 days and pH controlled at 6 ± 0.2. The highest level of caproate (4.8 g L-1) was observed at OLR of 15 gCOD L-1d-1 with a microbiome mainly composed by lactate-producing Actinomyces, Atopobium, and Olsenella species and caproate-producing Pseudoramibacter. Metagenomic analysis revealed the presence of key enzymes for the production of lactate, such as lactate dehydrogenase and pyruvate ferredoxin oxidoreductase, as well as several enzymes involved in the reverse ß-oxidation pathway, thus suggesting the occurrence of a lactate-based chain elongation process.

Investigating the influences of quorum quenching and nutrient conditions on activated sludge flocs at a short-time scale.

Quorum sensing signals regulate various functions within activated sludge processes such as formation of microbial aggregates. Disturbance of this signaling system, known as quorum quenching (QQ), provides opportunities for eliminating some problems related to biological wastewater treatment (e.g., biofouling and excess sludge production). However, it is poorly understood how and to what extent QQ systems can affect the microbial aggregation processes and the following floc formation. In particular, an in-depth structural characterization at the scale of microbial aggregate while considering nutrient conditions in the reactor is still largely disregarded. Here, we evaluated the QQ effects at the short-term time scale (i.e., after 4 h for the exogenous period and 19 h for exogenous/endogenous period), by combining advanced techniques for microbial characterization (flow cytometry, CARD-FISH, and confocal laser scanning microscopy) and conventional physical-chemical assessments. The results indicated that by implementing QQ agents (immobilized Acylase I enzyme in porous alginate beads) the abundance of single cells and suspended microbial aggregates in the supernatant did not show significant changes during the exogenous period. Conversely, at the end of the exogenous/endogenous period a significant increase of single prokaryotic cells, small and large microbial aggregates favored the growth of grazers, including free-living nanoflagellates and ciliates. Flocs became looser and thinner than those in the control reactor, thus affecting the sludge settling behavior. Inability of microbial community in degradation of soluble protein during the endogenous period confirmed that the QQ agents are likely to inhibit the secretion of protease enzyme within microbial communities of activated sludge.

Kitchen waste valorization through a mild-temperature pretreatment to enhance biogas production and fermentability: Kinetics study in mesophilic and thermophilic regimen.

Biowaste valorization through anaerobic digestion is an attractive option to achieve both climate protection goals and renewable energy production. In this paper, a complete set of batch trials was carried out on kitchen waste to investigate the effects of mild thermal pretreatment, temperature regimen and substrate/inoculum ratio. Thermal pretreatment was effective in the solubilisation of macromolecular fractions, particularly carbohydrates. The ability of the theoretical methodologies in estimating hydrogen and methane yields of complex substrates was evaluated by comparing the experimental results with the theoretical values. Despite the single batch configuration, a significant initial hydrogen production was observed, prior to methane yield. Main pretreatment effect was the gain in hydrogen production; the extent was highly variable according to the other parameters values. High hydrogen yields, up to 113 mL H2/g VSfed, were related to the prompt transformation of soluble sugars. Thermophilic regimen resulted, as expected, in faster digestions (up to 78 mL CH4/gVS/day) and sorted out pH inhibition. The relatively low methane yields (342-398 mL CH4/g VSfed) were the result of the consistent lignocellulosic content and low lipid content. Thermal pretreatment proved to be a promising option for the enhancement of hydrogen production in food waste dark fermentation.

Anaerobic digestion of mixed urban biowaste: The microbial community shift towards stability.

Anaerobic digestion is applied worldwide to treat food waste (FW) with the aim of obtaining renewable bioenergy by exploiting the methane gas produced. However, there are several problems in practical applications, primarily due to system instability. Although exhaustive knowledge regarding anaerobic microbial community composition has been established, few studies have investigated long-term correlations between microbial consortia, operative conditions and feedstock characteristics. Here, microbial community shifts as a response to feedstock variations were investigated in long-term semi-continuous systems, which were evaluated by an in situ cell detection method and 16S rRNA gene amplicon sequencing. FW digestion showed progressive system instability caused by the inhibition of methanogens, which resulted in volatile fatty acid accumulation and process failure at the low organic loading rate (OLR). Conversely, by co-digesting FW with waste-activated sludge (WAS), a stable process with methane yields of up to 0.27 Nm3 kg-1VSfed for OLR = 1.7 gVS L-1d-1 was achieved. This stabilizing effect was not related to the buffering capacity of WAS, but to its capacity to avoid volatile fatty acid accumulation and falls in pH by overcoming methanogenic activity inhibition. WAS addition promoted the establishment of a stable and active archaeal population in anaerobic co-digestion (AcoD) reactors. The continuous supply of trace elements together with the seeding of microbial functional groups were the main drivers that positively affected process stability.

Anaerobic co-digestion of food waste and waste activated sludge: ADM1 modelling and microbial analysis to gain insights into the two substrates' synergistic effects.

The reasons for the acidification problem affecting Food Waste (FW) anaerobic digestion were explored, combining the outcomes of microbiological data (FISH and CARD-FISH) and process modelling, based on the Anaerobic Digestion Model n°1 (ADM1). Long term semi continuous experiments were carried out, both with sole FW and with Waste Activated Sludge (WAS) as a co-substrate, at varying operational conditions (0.8-2.2 g VS L-1 d-1) and FW / WAS ratios. Acidification was observed along FW mono-digestion, making it necessary to buffer the digesters; ADM1 modelling and experimental results suggested that this phenomenon was due to the methanogenic activity decline, most likely related to a deficiency in trace elements. WAS addition, even at proportions as low as 10% of the organic load, settled the acidification issue; this ability was related to the promotion of the methanogenic activity and the consequent enhancement of acetate consumption, rather than to WAS buffering capacity. The ability of the ADM1 to model processes affected by low microbial activity, such as FW mono-digestion, was also assessed. It was observed that the ADM1 was only adequate for digestions with a high activity level for both bacteria and methanogens (FISH/CARD-FISH ratio preferably >0.8) and, under these conditions, the model was able to correctly predict the relative abundance of both microbial populations, extrapolated from FISH analysis.

Variability of food waste chemical composition: Impact of thermal pre-treatment on lignocellulosic matrix and anaerobic biodegradability.

A comprehensive sustainable Food Waste (FW) management is globally needed in order to reduce the environmental pollution and the financial costs due to FW disposal; anaerobic digestion is considered as one of the best environmental-friendly alternatives to this aim. A deep investigation of the chemical composition of different Food waste types (cooked kitchen waste (CKW), fruit and vegetable scraps (FVS) and organic fraction of municipal solid waste (OFMSW)) is here reported, in order to evaluate their relevant substance-specific properties and their impact on anaerobic biodegradability by means of a sophisticated automatic batch test system. Suitability for a mild thermal pre-treatment (T = 134 °C and p = 3.2 bar) to enhance the biological degradation of hardly accessible compounds was investigated. The pre-treatment affected significantly the carbohydrates solubilisation, and was able in reducing part of the lignocellulosic matrix. Moreover, in mesophilic conditions, the high solubilized sugars content favoured the initial recovery of hydrogen (not consumed by hydrogenotrophic methanogenesis), allowing to newly assess the extent of prompt fermentability. Pre-treatment enhanced hydrogen yields of FVS and OFMSW, with gains up to +50%, while the successive methane production, occurring in the same reactor, resulted affected by the lack of the soluble part of carbohydrates, "subtracted" for H2 production. Only in thermophilic conditions, when no hydrogen in the biogas was detected, pre-treatment of OFMSW significantly increased methane yield (from 0.343 to 0.389 L CH4 g-1 VSfed). A thermal pre-treatment seems the recommended solution in order to reduce part of the recalcitrant lignocellulosic matrix of food waste, to improve energy recovery and to eliminate the extra cost needed for pasteurization.

Enhanced Versus Conventional Sludge Anaerobic Processes: Performances and Techno-Economic Assessment.

Sewage sludge processing is a key issue in water resource recovery facilities due to the inefficacy of conventional treatments to produce high quality biosolids to be safely used in agriculture. Under this framework, the performances of several enhanced stabilization processes, namely ultrasound-pretreated Mesophilic Anaerobic Digestion (US+MAD), thermophilic anaerobic digestion (TAD), thermal-pretreated TAD (TH+TAD) and ultrasound-pretreated inverse Temperature Phased Anaerobic Digestion (US+iTPAD) have been investigated. Such enhanced processes resulted in higher biogas yields and higher destruction of pathogens with respect to conventional MAD process, thus suggesting their feasibility in full-scale implementation perspectives. A procedure for technical-economic comparison of new sludge processing lines against conventional ones (benchmarking) was developed, based on the definition of technical issues (e.g. reliability, complexity, etc.) which are rated for each situation. Moreover, capital and operating costs were estimated. The enhanced processes analyzed in this work showed some potentially critical items, mainly related to energy balance and reagent consumption.