Modelling of anaerobic digestion of microalgae biomass: Effect of overloading perturbation.

Anaerobic digestion (AD) of microalgae is an intriguing approach for bioenergy production. The scaling-up of AD presents a significant challenge due to the systematic efficiency losses related to process instabilities. To gain a comprehensive understanding of AD behavior, this study assessed a modified version of the anaerobic digestion model No1 (ADM1) + Contois kinetics to represent microalgae AD impacted by overloading. To this end, two new inhibition functions were implemented: inhibition by acetate for acidogenesis/acetogenesis and total volatile fatty acids for hydrolysis. This proposed ADM1 modification (including Contois kinetics) simulated AD behavior during the stable, disturbed and recovery periods, showing that the inhibition functions described in the original ADM1 cannot explain the AD performance under one of the most common perturbations at industrial scale (overloading). The findings underscore the importance of refining the inhibitions present in original ADM1 to better capture and predict the complexities of microalgae AD against overloading.

Effect of decoupling hydraulic and solid retention times on carbohydrate-rich residue valorization into carboxylic acids.

This research assessed the effect of decoupling hydraulic retention time (HRT) and solid retention time (SRT) on the production of volatile fatty acids (VFAs) via anaerobic fermentation of beet molasses. The performance of a continuous stirred tank reactor (CSTR, STR = HTR = 30 days) and two anaerobic sequencing batch reactors (AnSBR) with decoupled STR (30 days) and HRT (20 and 10 days) was compared. Previously, a temperature study in batch reactors (25, 35, and 55 °C) revealed 25 °C as the optimal temperature to maximize the VFAs yield and the long-chain VFAs (> C4) production, being selected for the continuous reactors operation. An HRT of 20 days in AnSBR led to an enhancement in bioconversion efficiency into VFAs (55.5% chemical oxygen demand basis) compared to the CSTR (34.9%). In contrast, the CSTR allowed the production of valuable caproic acid (25.4% vs 4.1% w/w of total VFAs in AnSBR). Decreasing further the HRT to 10 days in AnSBR was detrimental in terms of bioconversion efficiency (21.7%) due to primary intermediates (lactate) accumulation. By decoupling HRT and SRT, VFAs were maximized, revealing HRT as an effective tool to drive specific conversion routes (butyrate- or lactate-fermentation).

Hydraulic Retention Time as an Operational Tool for the Production of Short-Chain Carboxylates via Anaerobic Fermentation of Carbohydrate-Rich Waste.

The carboxylate platform is a sustainable and cost-effective way to valorize wastes into biochemicals that replace those of fossil origin. Short-chain fatty acids (SCFAs) are intermediates generated during anaerobic fermentation (AF) and are considered high-value-added biochemicals among carboxylates. This investigation aimed to produce SCFAs through the AF of sugar beet molasses at 25 °C and semi-continuous feeding mode in completely stirred tank reactors. A particular focus was devoted to the role of hydraulic retention time (HRT) variation in SCFAs production and distribution profile. The highest SCFAs concentration (44.1 ± 2.3 gCOD/L) was reached at the HRT of 30 days. Caproic acid accounted for 32.5-35.5% (COD-concentration basis) at the long HRTs of 20 and 30 days due to the carbon chain elongation of shorter carboxylic acids. The findings of this study proved that HRT could be used to steer the anaerobic process toward the targeted SCFAs for specific uses. Furthermore, the successful operation at low-temperature conditions (i.e., 25 °C) makes the process economically promising.

Microbial co-cultures for biochemicals production from lignocellulosic biomass: A review.

Global reliance on fossil oil should shift to cleaner alternatives to get a decarbonized society. One option to achieve this ambitious goal is the use of biochemicals produced from lignocellulosic biomass (LCB). The inherent low biodegradability of LCB and the inhibitory compounds that might be released during pretreatment are two main challenges for LCB valorization. At microbiological level, constraints are mostly linked to the need for axenic cultures and the preference for certain carbon sources (i.e., glucose). To cope with these issues, this review focuses on efficient LCB conversion via the sugar platform as well as an innovative carboxylate platform taking advantage of the co-cultivation of microorganisms. This review discusses novel trends in the use of microbial communities and co-cultures aiming at different bioproducts co-generation in single reactors as well as in sequential bioprocess combination. The outlook and further perspectives of these alternatives have been outlined for future successful development.

Influence of free ammonia extraction in methane production from human urine.

Human urine has a high chemical oxygen demand (COD) content which makes anaerobic treatments potentially appropriate for the management of yellow waters, allowing for energy recovery. However, its high N content makes this treatment challenging. The present work studied the viability of performing an anaerobic digestion process for COD valorization on a real (not synthetic) urine stream at laboratory scale. To deal with nitrogen inhibition, two different ammonia extraction systems were proposed and tested. With them, a proper evolution of acidogenesis and methanogenesis was observed. Nitrogen was recovered in the form of ammonium sulphate, which could be used for agriculture, in two different ways: ammonia extraction from the urine stream before feeding the reactor and in situ extraction in the reactor. The first method, which proved to be a better strategy consisted in a desorption process (NaOH addition, air bubbling and acid (H2SO4) absorption column, HCl for final pH adjustment) whereas the in situ extraction in the reactor consisted of an acid (H2SO4) absorption column installed in the biogas recycling line of both reactors. Stable methane production over 220 mL/g COD was achieved and methane content in the biogas was stable around 71%.

Production of short-chain fatty acids (SCFAs) as chemicals or substrates for microbes to obtain biochemicals.

Carboxylic acids have become interesting platform molecules in the last years due to their versatility to act as carbon sources for different microorganisms or as precursors for the chemical industry. Among carboxylic acids, short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, valeric, and caproic acids can be biotechnologically produced in an anaerobic fermentation process from lignocellulose or other organic wastes of agricultural, industrial, or municipal origin. The biosynthesis of SCFAs is advantageous compared to chemical synthesis, since the latter relies on fossil-derived raw materials, expensive and toxic catalysts and harsh process conditions. This review article gives an overview on biosynthesis of SCFAs from complex waste products. Different applications of SCFAs are explored and how these acids can be considered as a source of bioproducts, aiming at the development of a circular economy. The use of SCFAs as platform molecules requires adequate concentration and separation processes that are also addressed in this review. Various microorganisms such as bacteria or oleaginous yeasts can efficiently use SCFA mixtures derived from anaerobic fermentation, an attribute that can be exploited in microbial electrolytic cells or to produce biopolymers such as microbial oils or polyhydroxyalkanoates. Promising technologies for the microbial conversion of SCFAs into bioproducts are outlined with recent examples, highlighting SCFAs as interesting platform molecules for the development of future bioeconomy.

Genome-centric metagenomics revealed the effect of pH on the microbiome involved in short-chain fatty acids and ethanol production.

Added-value chemicals production via food waste (FWs) valorization using open-mixed cultures is an emerging approach to replace petrochemical-based compounds. Nevertheless, the effects of operational parameters on the product spectrum remain uncertain given the wide number of co-occurring species and metabolisms. In this study, the identification of 58 metagenome-assembled genomes and their investigation assessed the effect of slight pH variations on microbial dynamics and the corresponding functions when FWs were subjected to anaerobic fermentation (AF) in 1-L continuous stirred tank reactors at 25 °C. The initial pH of 6.5 promoted a microbial community involved in acetate, butyrate and ethanol production, mediated by Bifidobacterium subtile IE007 and Eubacteriaceae IE027 as main species. A slight pH decrease to 6.1 shaped microbial functions that resulted in caproate and H2 production, increasing the relevance of Eubacteriaceae IE037 role. This study elucidated the strong pH effect on product outputs when minimal variations take place in AF.

Microalgae production for nitrogen recovery of high-strength dry anaerobic digestion effluent.

Dry anaerobic digestion (D-AD) generates nitrogen-rich effluents that are normally neglected in the circular bioeconomy. The high turbidity and ammonium content hamper nitrogen recovery from these effluents via biological processes, such as microalgae culture. The goal of this study was to demonstrate microalgae growth viability in high-strength D-AD effluents in order to recover nitrogen (N) as microalgae biomass. According to the experimental factorial design conducted in batch reactors, ammonium was identified as the critical inhibitory compound for microalgae growth while turbidity did not exhibit a significantly negative effect. Instead, turbidity resulted advantageous since it promoted high nitrogen uptake rates and biomass production. The presence of organic turbidity resulted in a positive effect that boosted Chlorella growth in a stream with higher ammonium (350 mg NH4+-N L-1) and turbidity (175 NTU) than the inhibition thresholds reported in the literature, reaching 98.7% of N recovery as microalgae biomass. When microalgae culture was scaled up in a photobioreactor operated in continuous mode, microalgae biomass was effectively produced while recovering 100% of N at a hydraulic retention time of 10 days. By imposing long exposure times and high turbidity, Chlorella adaptation to high-strength D-AD effluent resulted in high N uptake and biomass production. This study demonstrated not only the most influencing factor and the optimal NH4+-N and turbidity combination, but also the viability of using D-AD effluents as culture media for microalgae biomass production.

Statistical correlation between waste macromolecular composition and anaerobic fermentation temperature for specific short-chain fatty acid production.

To properly exploit short-chain fatty acids (SCFAs) in the chemical industry, it is of foremost importance to ensure stable SCFA profile production via anaerobic fermentation (AF). The different macromolecular distribution of food wastes (FWs) used as feedstock might be crucial for process outcome. Targeting at a specific SCFAs profile and yield, this study explored the statistical correlation between the macromolecular composition of FWs and the produced SCFAs in batch-AFs at 25 °C and 55 °C. Principal Component Analysis (PCA) revealed that the carbohydrates fraction was directly related with butyric acid accumulation, regardless of process temperature. Nevertheless, operational temperature resulted in a pH change, which ultimately affected the process fate. PCA of 25 °C-batch-AF showed a positive correlation between high carbohydrate content and longer-chain acids accumulation. By contrast, 55 °C-AF resulted in higher product specificity than at 25 °C, mainly due to butyrate-type fermentation of carbohydrates. Batch results were further validated in a semicontinuous reactor. Prevailing SCFAs and high bioconversion efficiencies relied on 3 main FWs characteristics: high carbohydrate content (>77% w/w), high carbohydrate/protein ratio (≥10) and high soluble organic matter content. Results obtained herein allowed predicting a specific SCFAs profile based on FWs composition, which is relevant for setting proper downstream technologies.

Carboxylic acids production via anaerobic fermentation: Microbial communities' responses to stepwise and direct hydraulic retention time decrease.

Carboxylic acids, traditionally produced from fossil fuels, might be generated from renewable biomass resources via anaerobic fermentation. Considering that the microbial activity of this bioprocess is ruled by the imposed hydraulic retention time (HRT), this investigation explored the relationship between process stability and microbial community. Stepwise and direct HRT reduction strategies were assessed in terms of waste bioconversion into volatile fatty acids (VFAs). Microbial community dynamics revealed a microbial specialization along the HRT decrease. The direct implementation of low HRT resulted in drastic microbial fluctuations, leading to process failure at HRT below 6 days. Stepwise strategy for HRT reduction favored microbial adaptation, supporting maximum bioconversions efficiencies (32 % VFACOD/tCODin) at low HRT values (HRT 4 days). Microbial similarity analysis revealed Clostridiales, Lactobacillales and Bacteroidales orders as keystone microorganisms involved in VFAs production, being responsible for protein degradation and propionic acid accumulation.

Tuning microbial community in non-conventional two-stage anaerobic bioprocess for microalgae biomass valorization into targeted bioproducts.

Unspecific microorganisms consortia are normally used in anaerobic biodegradation of solid wastes. However, these consortia can be tuned to optimally obtain determined bioproducts. In this study, high value-added products and biogas were obtained via an innovative two-stage anaerobic bioprocess from microalgae biomass. The anaerobic fermentation (AF) entailed the production of short-chain fatty acids (SCFAs) and subsequently, only the solid spent of AF effluent was valorized for methane production via conventional anaerobic digestion (AD). Applied conditions in AF (25 °C, HRT 8 days) favored Firmicutes predominance (64%) enabling a conversion efficiency of 32.1% g SCFAs-COD/g CODin. Opposite, a wider microbial biodiversity was determined in the AD reactor (35 °C, HRT 20 days), being mainly composed by Firmicutes (28.6%), Euryarchaeota (17.7%) and Proteobacteria (15.3%). AD of the AF-solid spent reached 168.9 mL CH4 /g CODin. Strikingly, operational conditions imposed mediated a microbial specialization that maximized product output.

Valorisation of bioethanol production residues through anaerobic digestion: Methane production and microbial communities.

The organic fraction of municipal solid waste (OFMSW) is an appealing feedstock for bioethanol production due to its richness in cellulosic materials. After fermentation and distillation, the remaining residue constitutes a source of unconsumed carbohydrates, proteins and lipids. These macromolecules can be further used via anaerobic digestion (AD) for bioenergy purposes to offset bioethanol production costs. The present study evaluated the methanogenic potential of the whole fermented residue from a selective collection of OFMSW in a semicontinuous AD at 35 °C (HRT 40 days and OLR 2.09 g VS/Ld). The experimental results showed a methanogenic yield of 212 ± 5 mL CH4/g VSin (corresponding to a COD removal of 47 ± 1 %). Microbial analysis revealed key roles of species belonging to Firmicutes (65 %), Bacteroidetes (25 %) and Euryarchaeota (0.5-1 %). Methanosarcina archaea was highlighted as a robust methanogen crucial for methane production in a process in which the stability might be compromised by potential NH4+-N and VFAs inhibitions. This study indicated that the sequential combination of these two biochemical processes (fermentation and anaerobic digestion) allow to further exploit organic residues for their conversion into a marketable bioenergy product.

Life cycle assessment of volatile fatty acids production from protein- and carbohydrate-rich organic wastes.

Volatile fatty acids (VFAs) are platform molecules with numerous applications. They can be obtained by adjusting the operational conditions of anaerobic digestion to avoid methanogenesis while focusing on fermentative stages. There are gaps in the knowledge of how, from a life-cycle perspective, the fermentative process performs in VFAs production from waste, including environmental consequences of substituting common commodities in the current market. Mass and energy balances of VFAs production from protein-rich microalgal and carbohydrate-rich agro-industrial wastes were used herein as a key source of inventory data for life cycle assessment. Two waste treatment options were considered: (i) VFAs production (anaerobic fermentation) plus anaerobic digestion of the resulting waste after VFAs separation, and (ii) anaerobic digestion of the original waste for bioenergy. Several scenarios were formulated to evaluate their life-cycle performance. VFAs production generally shows a better environmental behaviour than conventional anaerobic digestion, principally due to the substitution of conventional chemicals.

Insights on the microbial communities developed during the anaerobic fermentation of raw and pretreated microalgae biomass.

Short-chain fatty acids (SCFAs) are considered building blocks for bioproducts in the so-called carboxylate platform. These compounds can be sustainably produced via anaerobic fermentation (AF) of organic substrates, such as microalgae. However, SCFAs bioconversion efficiency is hampered by the hard cell wall of some microalgae. In this study, one thermal and two enzymatic pretreatments (carbohydrases and proteases) were employed to enhance Chlorella vulgaris biomass solubilization prior to AF. Pretreated and non-pretreated microalgae were assessed in continuous stirred tank reactors (CSTRs) for SCFAs production. Aiming to understand microorganisms' roles in AF depending on the employed substrate, not only bioconversion yields into SCFAs were evaluated but microbial communities were thoroughly characterized. Proteins were responsible for the inherent limitation of raw biomass conversion into SCFAs. Indeed, the proteolytic pretreatment resulted in the highest bioconversion (33.4% SCFAs-COD/CODin), displaying a 4-fold enhancement compared with raw biomass. Population dynamics revealed a microbial biodiversity loss along the AF regardless of the applied pretreatment, evidencing that the imposed operational conditions specialized the microbial community. In fact, a reduced abundance in Euryarchaeota phylum explained the low methanogenic activity, implying SCFAs accumulation. The bacterial community developed in the reactors fed with pretreated microalgae exhibited high acidogenic activities, being dominated by Firmicutes and Bacteroidetes. Firmicutes was by far the dominant phylum when using protease (65% relative abundance) while Bacteroidetes was prevailing in the reactor fed with carbohydrase-pretreated microalgae biomass (40% relative abundance). This fact indicated that the applied pretreatment and macromolecule solubilization have a strong effect on microbial distribution and therefore in SCFAs bioconversion yields.

Anaerobic degradation of protein-rich biomass in an UASB reactor: Organic loading rate effect on product output and microbial communities dynamics.

Anaerobic degradation of enzymatically pretreated Chlorella vulgaris was aimed in an upflow anaerobic sludge blanket reactor (UASB) to evaluate the organic loading rate (OLR) effect on biomass valorization. Low OLRs resulted in high methane yields (171 mL CH4/g CODin) at low hydraulic retention time (HRT of 6 days). Firmicutes (35-43%), Bacteroidetes (17-18%) and Euryarchaeota (11%) dominated at low OLRs, promoting methanogenic activity. On the contrary, the highest OLRs resulted in low methane yield (86 mL CH4/gCODin) with a concomitant short-chain fatty acids (SCFAs) accumulation of 37% SCFAs-COD/CODin. The highest OLR decreased UASB reactor biodiversity, hampering Euryarchaeota population development (2.5%) and boosting Firmicutes (55%) and Proteobacteria (14%). These results demonstrated the suitability of UASB reactor configuration to reach high bioprocess efficiency for both, biogas and SCFAs production, with lower energetic and area requirements than those normally needed in continuous stirred tank reactors.

Agroindustrial waste as a resource for volatile fatty acids production via anaerobic fermentation.

This study evaluated the feasibility of the anaerobic digestion as a sustainable valorisation strategy for volatile fatty acids production from agroindustrial waste (cucumber, tomato and lettuce). High bioconversion efficiencies were reached by operating the reactors at 25 °C, 3 g VS·d-1·L-1 with pH adjustment. Cucumber fermentation achieved the highest bioconversion (52.6%), whereas tomato degradation was the least efficient bioprocess (40.1%) due to the low pH (5.6) that partially inhibited the hydrolytic and acidogenic activities. In all cases, carboxylic acid profiles were mainly composed of volatile fatty acids with even carbon number. The developed microbial community exhibited high hydrolytic and acidogenic activities associated to carbohydrates degradation. This microbial population was dominated by Firmicutes phylum and showed a lack of acetogenic bacteria related with CH4 production, resulting in a remarkably high VFAs accumulation.

Impact of Organic Loading Rate in Volatile Fatty Acids Production and Population Dynamics Using Microalgae Biomass as Substrate.

Volatile fatty acids (VFAs) are regarded as building blocks with a wide range of applications, including biofuel production. The traditional anaerobic digestion used for biogas production can be alternatively employed for VFAs production. The present study aimed at maximizing VFAs productions from Chlorella vulgaris through anaerobic digestion by assessing the effect of stepwise organic loading rates (OLR) increases (3, 6, 9, 12 and 15 g COD L-1 d-1). The biological system was proven to be robust as organic matter conversion efficiency into VFAs increased from 0.30 ± 0.02 COD-VFAs/CODin at 3 g COD L-1 d-1 to 0.37 ± 0.02 COD-VFAs/CODin at 12 g COD L-1d-1. Even though, the hydrolytic step was similar for all studied scenario sCOD/tCOD = 0.52-0.58), the highest OLR (15 g COD L-1 d-1) did not show any further increase in VFAs conversion (0.29 ± 0.01 COD-VFAs/CODin). This fact suggested acidogenesis inhibition at 15 g COD L-1d-1. Butyric (23-32%), acetic (19-26%) and propionic acids (11-17%) were the most abundant bioproducts. Population dynamics analysis revealed microbial specialization, with a high presence of Firmicutes followed by Bacteroidetes. In addition, this investigation showed the microbial adaptation of Euryarchaeota species at the highest OLR (15 g COD L-1d-1), evidencing one of the main challenges in VFAs production (out-competition of archaea community to avoid product consumption). Stepwise OLR increase can be regarded as a tool to promote VFAs productions. However, acidogenic inhibition was reported at the highest OLR instead of the traditional hydrolytic barriers. The operational conditions imposed together with the high VFAs and ammonium concentrations might have affected the system yields. The relative abundance of Firmicutes (74%) and Bacteroidetes (20%), as main phyla, together with the reduction of Euryarchaeota phylum (0.5%) were found the best combination to promote organic matter conversion into VFAs.