Bayesian uncertainty quantification for anaerobic digestion models.

Uncertainty quantification is critical for ensuring adequate predictive power of computational models used in biology. Focusing on two anaerobic digestion models, this article introduces a novel generalized Bayesian procedure, called VarBUQ, ensuring a correct tradeoff between flexibility and computational cost. A benchmark against three existing methods (Fisher's information, bootstrapping and Beale's criteria) was conducted using synthetic data. This Bayesian procedure offered a good compromise between fitting ability and confidence estimation, while the other methods proved to be repeatedly overconfident. The method's performances notably benefitted from inductive bias brought by the prior distribution, although it requires careful construction. This article advocates for more systematic consideration of uncertainty for anaerobic digestion models and showcases a new, computationally efficient Bayesian method. To facilitate future implementations, a Python package called 'aduq' is made available.

Resource recovery using enriched purple phototrophic bacteria in an outdoor flat plate photobioreactor: Suspended vs. attached growth.

Purple phototrophic bacteria (PPB) can produce single-cell protein from wastewater at high yields. Growing in a biofilm vs suspended can improve product quality and consistency. This study compares suspended and attached growths of enriched PPB cultures in an outdoor flat plate photobioreactor treating poultry-processing wastewater. Attached growth had lower VFA removal efficiencies (95 ± 2.7 vs 84 ± 6.4 %) due to light limitations and low substrate diffusion rates. Nevertheless, similar overall treatment performances and productivities were achieved (16 ± 2.2 and 18 ± 2.4 gCOD·m-2·d-1 for attached and suspended) at loading rates of 1.2-1.5 gCOD·L-1·d-1. Biofilms had higher quality than suspended biomass, with lower ash contents (6.9(0.6)% vs 57(16)%) and higher PPB abundances (0.45-0.67 vs 0.30-0.45). The biofilm (20-50 % of the total biomass) might be used as feed and the suspended fraction as fertiliser, improving the economics of the process. Semi-continuous PPB growth outdoors as biofilm is technically feasible, obtaining a superior product without jeopardising performance.

Expanding mechanistic models to represent purple phototrophic bacteria enriched cultures growing outdoors.

The economic feasibility of purple phototrophic bacteria (PPB) for resource recovery relies on using enriched-mixed cultures and sunlight. This work presents an extended Photo-Anaerobic Model (ePAnM), considering: (i) the diverse metabolic capabilities of PPB, (ii) microbial clades interacting with PPB, and (iii) varying environmental conditions. Key kinetic and stoichiometric parameters were either determined experimentally (with dedicated tests), calculated, or gathered from literature. The model was calibrated and validated using different datasets from an outdoors demonstration-scale reactor, as well as results from aerobic and anaerobic batch tests. The ePAnM was able to predict the concentrations of key compounds/components (e.g., COD, volatile fatty acids, and nutrients), as well as microbial communities (with anaerobic systems dominated by fermenters and PPB). The results underlined the importance of considering other microbial clades and varying environmental conditions. The model predicted a minimum hydraulic retention time of 0.5 d-1. A maximum width of 10 cm in flat plate reactors should not be exceeded. Simulations showed the potential of a combined day-anaerobic/night-aerobic operational strategy to allow efficient continuous operation.

Method development for PPB culture screening, pigment analysis with UPLC-UV-HRMS vs. spectrophotometric methods, and spectral decomposition-based analysis.

PPB carotenoids are usually measured through spectrophotometric analysis, measuring total carotenoids (TCs) which has low accuracy and cannot identify individual carotenoids or isomers. Here, we developed an ultra-performance liquid chromatography method with ultraviolet and high-resolution mass spectrometry detection (UPLC-UV-HRMS) to quantify neurosporene, lycopene, and bacteriochlorophyll a contents in PPB cultures. The method exhibited satisfactory recoveries for individual pigments (between 82.1% and 99.5%) and was applied to a range of mixed PPB cultures. The use of a C30 column also enabled the detection of three different isomers of lycopene. In addition, a method for anaerobic photoheterotrophic PPB cultivation to acquire live-cell spectrophotometric information was developed and tested by modifying a standard microbial culture microplate system. A rapid, and relatively low effort principal component analysis (PCA) based decomposition of the whole-cell spectra for pigment analysis in the microplates was also developed. Analysing whole-cell spectra via PCA allowed more accurate prediction of individual pigments compared to absorption methods, and can be done non-destructively, during live-cell growth, but requires calibration for new media and microbial matrices.

Creating value from purple phototrophic bacteria via single-cell protein production.

Mixed culture purple phototrophic bacteria (PPB) is a rapidly emerging technology for resource recovery from wastewaters. PPB biomass can be used as single-cell protein, with a high protein content complemented by value-added components (e.g. pigments and polyhydroxyalkanoates), merging functionalities within a single product. This has the potential to increase the value and impact the economic feasibility, justifying higher capital costs for PPB photobioreactors for real-life applications. Artificial illumination is prohibitively expensive, and naturally illuminated, outdoor units are a critical next step. However, information required for informed technoeconomic assessment of single-cell protein from PPB is still missing and can only be determined in dedicated larger-scale, outdoor systems. Larger scale units are also required to supply feed for larger cohort trials. Although data from microalgae research can be used as starting point to estimate costs, they cannot be translated directly for PPB, as the organisms and metabolic growth are fundamentally different.

Light attenuation in enriched purple phototrophic bacteria cultures: Implications for modelling and reactor design.

Light attenuation in enriched purple phototrophic bacteria (PPB) cultures has not been studied, and its understanding is critical for proper process modelling and reactor design, especially for scaled systems. This work evaluated the effect of different biomass concentrations, reactor configurations, wastewater matrices, and growth conditions, on the attenuation extent of near infra-red (NIR) and ultraviolet-visible (UV-VIS) light spectra. The results show that increased biomass concentrations lead to higher light attenuation, and that PPB absorb both VIS and NIR wavelengths, with both fractions of the spectrum being equally absorbed at biomass concentrations above 1,000 g COD·m-3. A flat plate configuration showed less attenuation compared with cylindrical reactors illuminated from the top, representative for open ponds. Neither a complex wastewater matrix nor the presence of polyhydroxyalkanoates (under nutrient limited conditions) affected light attenuation significantly. The pigment concentration (both bacteriochlorophyll and carotenoids) however, had a strong effect, with significant attenuation in the presence of pigments. Attenuation predictions using the Lambert-Beer law (excluding scattering) and the Schuster model (including scattering) indicated that light scattering had a minimal effect. A proposed mathematical model, based on the Lambert-Beer law and a Monod function for light requirements, allowed effective prediction of the kinetics of photoheterotrophic growth. This resulted in a half saturation coefficient of 4.6 W·m-2. Finally, the results showed that in dense outdoor PPB cultures (≥1,000 g COD·m-3), effective light penetration is only 5 cm, which biases design away from horizontal lagoons, and towards non-incident multi-panel systems such as flat plate reactors.

Outdoor demonstration-scale flat plate photobioreactor for resource recovery with purple phototrophic bacteria.

To make purple phototrophic bacteria (PPB)-based technologies a reality for resource recovery, research must be demonstrated outdoors, using scaled reactors. In this study, a 10 m long PPB-enriched flat plate photobioreactor (FPPBR) with a volume of 0.95 m3 was operated for 253 days, fed with poultry processing wastewater. Different operational strategies were tested, including varying influent types, retention times, feeding strategies, and anaerobic/aerobic conditions in a novel mixed metabolic mode concept. The overall results show that regardless of the fermented wastewater fed (raw or after solid removal via dissolved air flotation) and the varying environmental conditions (e.g., light exposure and temperatures), the FPPBR provided effective volatile fatty acids (VFAs), N, and P removals (average efficiencies of >90%, 34-77%, and 28-45%, respectively). The removal of N and P was limited by the availability of biodegradable COD. Biomass (C, N and P) could be harvested at ∼90% VS/TS ratio, 58% crude protein content and a suitable amino acid profile for potential feed applications. During fully anaerobic operation with semicontinuous/day-only feeding, the FPPBR showed biomass productivities between 25 and 84 g VS m-2 d-1 (high due to solid influx; the productivities estimated from COD removal rates were 6.0-24 g VS•m-2•d-1 (conservative values)), and soluble COD removal rates of up to 1.0 g•L-1•d-1 (overall average of 0.34 ± 0.16 g•L-1•d-1). Under these conditions, the relative abundance of PPB in the harvested biomass was up to 56%. A minimum overall HRT of 2-2.4 d (1.0-1.2 d when only fed during the day) is recommended to avoid PPB washout, assuming no biomass retention. A combined daily-illuminated-anaerobic/night-aerobic operation (supplying air during night-time) exploiting photoheterotrophy during the day and aerobic chemoheterotrophy of the same bacteria at night improved the overall removal performance, avoiding VFA accumulation during the night. However, while enabling enhanced treatment, this resulted in a lower relative abundance of PPB and reduced biomass productivities, highlighting the need to balance resource recovery and treatment goals.

Naturally illuminated photobioreactors for resource recovery from piggery and chicken-processing wastewaters utilising purple phototrophic bacteria.

Resource recovery from wastewater, preferably as high value products, has become an integral part of modern wastewater treatment. This work presents the potential to produce single cell protein (SCP) from pre-settled piggery wastewater (PWW) and meat chicken processing wastewater (CWW), utilising anaerobic purple phototrophic bacteria (PPB). PPB were grown as biofilm in outdoors 60 L, 80 L and 100 L flat-plate reactors, operated in sequential batch mode. PPB biofilm was recovered from reactor walls at a total solid (TS) content ∼90 g•L - 1, and the harvested biomass (depending on the wastewater) had a consistent quality, with high protein contents (50-65%) and low ash, potentially applicable as SCP. The COD, N and P removal efficiencies were 71±5.3%, 22±6.6%, 65±5.6% for PWW and 78±1.8%, 67±2.7% and 37±4.0% for CWW, respectively, with biofilm areal productivities up to 14 g TS•m - 2•d - 1. This was achieved at ammonium-N concentrations over 1.0 g•L - 1 and temperatures up to 55 °C and down to 6 °C (daily fluctuations of 20-30 °C). The removal performances and biomass productivities were mostly dependent on the bioavailable COD in the form of volatile fatty acids (VFA). At sufficient VFA availability, the irradiance became limiting, capping biofilm formation. Harvesting of the suspended fraction resulted in increased productivities and recovery efficiencies, but lowered the product quality (e.g., containing undesired inerts). The optimum between quantity and quality of product is dependent on the wastewater characteristics (i.e., organic degradable fraction) and potential pre-treatment. This study shows the potential to utilise sunlight to treat agri-industrial wastewaters while generating protein-rich PPB biomass to be used as a feed, feed additive or feed supplement.

The interaction between lipids and ammoniacal nitrogen mitigates inhibition in mesophilic anaerobic digestion.

Ammoniacal nitrogen and long chain fatty acids (LCFA) are common inhibitors of the anaerobic digestion process. However, the interaction between these inhibitors has received little attention. Understanding the interaction between these inhibitors is important to optimise the operation of anaerobic digesters treating slaughterhouse waste or using fat, oil and grease (FOG) as co-substrate among others. To study the interaction between ammoniacal nitrogen and LCFA inhibition, 20 different conditions were trialled in mesophilic batch tests. Experimental conditions included 5 mixtures between slaughterhouse wastewater and LCFA (100:0, 75:25, 50:50, 20:80, 0:100 on a VS basis), each one tested at 4 different ammoniacal nitrogen concentrations (0, 1, 3, 6 gNadded·L-1). Experimental and modelling results showed that ammoniacal nitrogen inhibition was less severe in LCFA-rich mixtures, indicating that LCFA mitigated ammoniacal nitrogen inhibition to a certain extent. However, the positive interaction between inhibitors did not only depend on the LCFA concentration. A protective LCFA coat that limited the diffusion of free ammonia into the cell and/or provided a localised lower pH in the vicinity of the microbial cell could explain the experimental results. However, ammoniacal nitrogen and LCFA inhibition comprise up to 6 different but interrelated inhibitors (i.e. NH3, NH4+, LCFA, VFA, H2 and pH) and therefore the specific mechanism could not be elucidated. Nonetheless, these results suggest that LCFA do not exacerbate TAN-related inhibition and that LCFA-rich substrates can be utilised as co-substrates in mesophilic N-rich digesters.

Considering syntrophic acetate oxidation and ionic strength improves the performance of models for food waste anaerobic digestion.

Current mechanistic anaerobic digestion (AD) models cannot accurately represent the underlying processes occurring during food waste (FW) AD. This work presents an update of the Anaerobic Digestion Model no. 1 (ADM1) to provide accurate estimations of free ammonia concentrations and related inhibition thresholds, and model syntrophic acetate oxidation as acetate-consuming pathway. A modified Davies equation predicted NH3 concentrations and pH more accurately, and better estimated associated inhibitory limits. Sensitivity analysis results showed the importance of accurate disintegration kinetics and volumetric mass transfer coefficients, as well as volatile fatty acids (VFAs) and hydrogen uptake rates. In contrast to the default ADM1, the modified ADM1 could represent methane production and VFA profiles simultaneously (particularly relevant for propionate uptake). The modified ADM1 was also able to predict the predominant acetate-consuming and methane-producing microbial clades. Modelling results using data from reactors dosed with granular activated carbon showed that this additive improves hydrogen uptake.

Purple phototrophic bacteria are outcompeted by aerobic heterotrophs in the presence of oxygen.

There is an ongoing debate around the effect of microaerobic/aerobic conditions on the wastewater treatment performance and stability of enriched purple phototrophic bacteria (PPB) cultures. It is well known that oxygen-induced oxidative conditions inhibit the synthesis of light harvesting complexes, required for photoheterotrophy. However, in applied research, several publications have reported efficient wastewater treatment at high dissolved oxygen (DO) levels. This study evaluated the impact of different DO concentrations (0-0.25 mg·L-1, 0-0.5 mg·L-1 and 0-4.5 mg·L-1) on the COD, nitrogen and phosphorus removal performances, the biomass yields, and the final microbial communities of PPB-enriched cultures, treating real wastewaters (domestic and poultry processing wastewater). The results show that the presence of oxygen suppressed photoheterotrophic growth, which led to a complete pigment and colour loss in a matter of 20-30 h after starting the batch. Under aerobic conditions, chemoheterotrophy was the dominant catabolic pathway, with wastewater treatment performances similar to those achieved in common aerobic reactors, rather than those corresponding to phototrophic systems (i.e. considerable total COD decrease (45-57% aerobically vs. ± 10% anaerobically). This includes faster consumption of COD and nutrients, lower nutrient removal efficiencies (50-58% vs. 72-99% for NH4+-N), lower COD:N:P substrate ratios (100:4.5-5.0:0.4-0.8 vs. 100:6.7-12:0.9-1.2), and lower apparent biomass yields (0.15-0.31 vs. 0.8-1.2 g CODbiomass·g CODremoved-1)). The suppression of photoheterotrophy inevitably resulted in a reduction of the relative PPB abundances in all the aerated tests (below 20% at the end of the tests), as PPB lost their main competitive advantage against competing aerobic heterotrophic microbes. This was explained by the lower aerobic PPB growth rates (2.4 d-1 at 35 °C) when compared to common growth rates for aerobic heterotrophs (6.0 d-1 at 20 °C). Therefore, PPB effectively outcompete other microbes under illuminated-anaerobic conditions, but not under aerobic or even micro-aerobic conditions, as shown by continuously aerated tests controlled at undetectable DO levels. While their aerobic heterotrophic capabilities provide some resilience, at non-sterile conditions PPB cannot dominate when growing chemoheterotrophically, and will be outcompeted.

Purple phototrophic bacteria for resource recovery: Challenges and opportunities.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a "removal and disposal" approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved-1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3-2.2 and 0.1-0.3 $·kgdry biomass-1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5-2.0 $·kg-1) appears as a promising valorisation route.

Microalgae-bacteria consortia in high-rate ponds for treating urban wastewater: Elucidating the key state indicators under dynamic conditions.

On-line performance indicators of a microalgae-bacteria consortium were screened out from different variables based on pH and dissolved oxygen on-line measurements via multivariate projection analysis, aiming at finding on-line key state indicators to easily monitor the process. To fulfil this objective, a pilot-scale high-rate pond for urban wastewater treatment was evaluated under highly variable conditions, i.e. during the start-up period. The system was started-up without seed of either bacterial or microalgal biomass. It took around 19 days to fully develop a microalgal community assimilating nutrients significantly. Slight increases in the biomass productivities in days 26-30 suggest that the minimum time for establishing a performant bacteria-microalgae consortium could be of around one month for non-inoculated systems. At this point, the process was fully functional, meeting the European discharge limits for protected areas. The results of the statistical analyses show that both the pH and the dissolved oxygen concentration represent accurately the biochemical processes taking place under the start-up of the process. Both pH and dissolved oxygen represented accurately also the performance of the high-rate algal pond, being affordable, easily-implemented, options for monitoring, control and optimization of industrial-scale processes.

Performance of a membrane-coupled high-rate algal pond for urban wastewater treatment at demonstration scale.

The objective of this study was to evaluate the performance of an outdoor membrane-coupled high-rate algal pond equipped with industrial-scale membranes for treating urban wastewater. Decoupling biomass retention time (BRT) and hydraulic retention time (HRT) by membrane filtration resulted in improved process efficiencies, with higher biomass productivities and nutrient removal rates when operating at low HRTs. At 6 days of BRT, biomass productivity increased from 30 to 66 and to 95 g·m-3·d-1 when operating at HRTs of 6, 4 and 2.5 days, respectively. The corresponding nitrogen removal rates were 4, 8 and 11 g N·m-3·d-1 and the phosphorous removal rates were 0.5, 1.3 and 1.6 g P·m-3·d-1. The system was operated keeping moderate specific air demands (0.25 m3·m-2·h-1), resulting in reasonable operating and maintenance costs (€0.04 per m3) and energy requirements (0.29 kWh per m3). The produced water was free of pathogens and could be directly used for reusing purposes.

Methanosarcina plays a main role during methanogenesis of high-solids food waste and cardboard.

Anaerobic digestion of food waste is a complex process often hindered by high concentrations of volatile fatty acids and ammonia. Methanogenic archaea are more sensitive to these inhibitors than bacteria and thus the structure of their community is critical to avoid reactor acidification. In this study, the performances of three different inocula were compared using batch digestion tests of food waste and cardboard mixtures. Particular attention was paid to the archaeal communities in the inocula and after digestion. While the tests started with inocula rich in Methanosarcina led to efficient methane production, VFAs accumulated in the reactors where inocula initially were poor in this archaea and no methane was produced. In addition, higher substrate loads were tolerated when greater proportions of Methanosarcina were initially present in the inoculum. Independently of the inoculum origin, Methanosarcina were the dominant methanogens in the digestates from the experiments that efficiently produced methane. These results suggest that the initial archaeal composition of the inoculum is crucial during reactor start-up to achieve stable anaerobic digestion at high concentrations of ammonia and organic acids.

Addition of granular activated carbon and trace elements to favor volatile fatty acid consumption during anaerobic digestion of food waste.

The effect of supplementing granular activated carbon and trace elements on the anaerobic digestion performance of consecutive batch reactors treating food waste was investigated. The results from the first batch suggest that addition of activated carbon favored biomass acclimation, improving acetic acid consumption and enhancing methane production. Adding trace elements allowed a faster consumption of propionic acid. A second batch proved that a synergy existed when activated carbon and trace elements were supplemented simultaneously. The degradation kinetics of propionate oxidation were particularly improved, reducing significantly the batch duration and improving the average methane productivities. Addition of activated carbon favored the growth of archaea and syntrophic bacteria, suggesting that interactions between these microorganisms were enhanced. Interestingly, microbial analyses showed that hydrogenotrophic methanogens were predominant. This study shows for the first time that addition of granular activated carbon and trace elements may be a feasible solution to stabilize food waste anaerobic digestion.

Cardboard proportions and total solids contents as driving factors in dry co-fermentation of food waste.

This study evaluated the influence of the co-substrate proportions (0-60% of cardboard in dry basis) and the initial total solid contents (20-40%) on the batch fermentation performance. Maximum hydrogen yields were obtained when mono-fermenting food waste at high solids contents (89mlH2·gVS-1). The hydrogen yields were lower when increasing the proportions of cardboard. The lower hydrogen yields at higher proportions of cardboard were translated into higher yields of caproic acid (up to 70.1gCOD·kgCODbio-1), produced by consumption of acetic acid and hydrogen. The highest substrate conversions were achieved at low proportions of cardboard, indicating a stabilization effect due to higher buffering capacities in co-fermentation. Clostridiales were predominant in all operational conditions. This study opens up new possibilities for using the cardboard proportions for controlling the production of high added-value products in dry co-fermentation of food waste.

Kinetic study of dry anaerobic co-digestion of food waste and cardboard for methane production.

Dry anaerobic digestion is a promising option for food waste treatment and valorization. However, accumulation of ammonia and volatile fatty acids often occurs, leading to inefficient processes and digestion failure. Co-digestion with cardboard may be a solution to overcome this problem. The effect of the initial substrate to inoculum ratio (0.25 to 1gVS·gVS-1) and the initial total solids contents (20-30%) on the kinetics and performance of dry food waste mono-digestion and co-digestion with cardboard was investigated in batch tests. All the conditions produced methane efficiently (71-93% of the biochemical methane potential). However, due to lack of methanogenic activity, volatile fatty acids accumulated at the beginning of the digestion and lag phases in the methane production were observed. At increasing substrate to inoculum ratios, the initial acid accumulation was more pronounced and lower cumulative methane yields were obtained. Higher amounts of soluble organic matter remained undegraded at higher substrate loads. Although causing slightly longer lag phases, high initial total solids contents did not jeopardize the methane yields. Cardboard addition reduced acid accumulation and the decline in the yields at increasing substrate loads. However, cardboard addition also caused higher concentrations of propionic acid, which appeared as the most last acid to be degraded. Nevertheless, dry co-digestion of food waste and cardboard in urban areas is demonstrated asan interesting feasible valorization option.

Accumulation of propionic acid during consecutive batch anaerobic digestion of commercial food waste.

The objective of this study was to test three different alternatives to mitigate the destabilizing effect of accumulation of ammonia and volatile fatty acids during food waste anaerobic digestion. The three options tested (low temperature, co-digestion with paper waste and trace elements addition) were compared using consecutive batch reactors. Although methane was produced efficiently (∼500ml CH4gVS-1; 16l CH4lreactor-1), the concentrations of propionic acid increased gradually (up to 21.6gl-1). This caused lag phases in the methane production and eventually led to acidification at high substrate loads. The addition of trace elements improved the kinetics and allowed higher substrate loads, but could not avoid propionate accumulation. Here, it is shown for the first time that addition of activated carbon, trace elements and dilution can favor propionic acid consumption after its accumulation. These promising options should be optimized to prevent propionate accumulation.

Dry anaerobic digestion of food waste and cardboard at different substrate loads, solid contents and co-digestion proportions.

The increasing food waste production calls for developing efficient technologies for its treatment. Anaerobic processes provide an effective waste valorization. The influence of the initial substrate load on the performance of batch dry anaerobic co-digestion reactors treating food waste and cardboard was investigated. The load was varied by modifying the substrate to inoculum ratio (S/X), the total solids content and the co-digestion proportions. The results showed that the S/X was a crucial parameter. Within the tested values (0.25, 1 and 4gVS·gVS-1), only the reactors working at 0.25 produced methane. Methanosarcina was the main archaea, indicating its importance for efficient methanogenesis. Acidogenic fermentation was predominant at higher S/X, producing hydrogen and other metabolites. Higher substrate conversions (≤48%) and hydrogen yields (≤62mL·gVS-1) were achieved at low loads. This study suggests that different value-added compounds can be produced in dry conditions, with the initial substrate load as easy-to-control operational parameter.