Waste-derived volatile fatty acids as carbon source for added-value fermentation approaches.

The establishment of a sustainable circular bioeconomy requires the effective material recycling from biomass and biowaste beyond composting/fertilizer or anaerobic digestion/bioenergy. Recently, volatile fatty acids attracted much attention due to their potential application as carbon source for the microbial production of high added-value products. Their low-cost production from different types of wastes through dark fermentation is a key aspect, which will potentially lead to the sustainable production of fuels, materials or chemicals, while diminishing the waste volume. This article reviews the utilization of a volatile fatty acid platform for the microbial production of polyhydroxyalkanoates, single cell oil and omega-3 fatty acids, giving emphasis on the fermentation challenges for the efficient implementation of the bioprocess and how they were addressed. These challenges were addressed through a research project funded by the European Commission under the Horizon 2020 programme entitled 'VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks'.

Interfacing anaerobic digestion with (bio)electrochemical systems: Potentials and challenges.

For over a century, anaerobic digestion has been a key technology in stabilizing organic waste streams, while at the same time enabling the recovery of energy. The anticipated transition to a bio-based economy will only increase the quantity and diversity of organic waste streams to be treated, and, at the same time, increase the demand for additional and effective resource recovery schemes for nutrients and organic matter. The performance of anaerobic digestion can be supported and enhanced by (bio)electrochemical systems in a wide variety of hybrid technologies. Here, the possible benefits of combining anaerobic digestion with (bio)electrochemical systems were reviewed in terms of (1) process monitoring, control, and stabilization, (2) nutrient recovery, (3) effluent polishing, and (4) biogas upgrading. The interaction between microorganisms and electrodes with respect to niche creation is discussed, and the potential impact of this interaction on process performance is evaluated. The strength of combining anaerobic digestion with (bio)electrochemical technologies resides in the complementary character of both technologies, and this perspective was used to distinguish transient trends from schemes with potential for full-scale application. This is supported by an operational costs assessment, showing that the economic potential of combining anaerobic digestion with a (bio)electrochemical system is highly case-specific, and strongly depends on engineering challenges with respect to full-scale applications.

Electrochemical Ammonia Recovery from Source-Separated Urine for Microbial Protein Production.

Conventional plant and meat protein production have low nitrogen usage efficiencies and high energy needs. Microbial protein (MP) is an alternative that offers higher nitrogen conversion efficiencies with low energy needs if nitrogen is recovered from a concentrated waste source such as source-separated urine. An electrochemical cell (EC) was optimized for ammonia recovery as NH3/H2 gas mixtures usable for MP production. Undiluted hydrolyzed urine was fed to the caustic-generating cathode compartment for ammonia stripping with redirection to the anode compartment for additional ammonium extraction. Using synthetic urine at 48 A m-2 the nitrogen removal efficiency reached 91.6 ± 2.1%. Tests with real urine at 20 A m-2, achieved 87.1 ± 6.0% and 68.4 ± 14.6% requiring 5.8 and 13.9 kWh kg N-1 recovered, via absorption in acid or MP medium, respectively. Energy savings through accompanying electrolytic H2 and O2 production were accounted for. Subsequently, MP was grown in fed-batch on MP medium with conventional NH4+ or urine-derived NH3 yielding 3.74 ± 1.79 and 4.44 ± 1.59 g CDW L-1, respectively. Dissolution of gaseous NH3 in MP medium maintained neutral pH in the MP reactor preventing caustic addition and thus salt accumulation. Urine-nitrogen could thus be valorized as MP via electrochemical ammonia recovery.

Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation.

BACKGROUND: The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures. RESULTS: Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n-caproic acid, which were up to 40 mM day-1 (4.64 g L-1 day-1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n-caproic acid production. In addition, n-caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions. CONCLUSIONS: Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.

A Gibbs Free Energy-Based Assessment of Microbial Electrocatalysis.

The use of microbial catalysts for electrode reactions enables novel bioremediation and bioproduction processes. To understand the electrochemical performance of the electrode reactions, knowledge of their thermodynamics is essential. We elaborate here on the Growth Reference System (GRS), simplifying thermodynamic calculations in the aforementioned context to, for example, demonstrate that cathodic bioprocesses generally suffer from higher overpotentials than do anodic processes. Abiotic hydrogen production cannot be thermodynamically excluded for any of the cathodic microbial electrosynthesis processes described thus far. Predictions for maximum biomass production correlated to electron flow are in line with experimental observations. We include a comprehensive set of thermodynamic and electrochemical data to support calculations relevant to the field of microbial electrocatalysis.

The type of ion selective membrane determines stability and production levels of microbial electrosynthesis.

Microbial electrosynthesis (MES) can enable electricity-driven bioproduction from CO2. Several membrane types such as anion exchange, cation exchange, and bipolar membranes (AEM/CEM/BPM) can be used to separate the anodic oxidation from the biocathodic reduction. The impact of the membrane type on MES has not yet been studied. Therefore we compared the three membranes for MES of acetic acid. The reactor with AEM enabled in situ recovery of acetic acid. This extraction led to a 32% higher production rate and efficiency compared to the systems that did not include product recovery, as product inhibition was likely occurring. Besides H+/OH-, mainly HCO3- contributed to charge balancing. Due to water displacement across the membrane, the product concentration in the AEM reactor (9g/L) did not exceed the concentration in the CEM reactor (10.5g/L). Overall this comparison shows that the membrane type in MES can be critical towards a stable and efficient process.

Extraction and Esterification of Low-Titer Short-Chain Volatile Fatty Acids from Anaerobic Fermentation with Ionic Liquids.

Ionic liquids can both act as a solvent and mediate esterification to valorize low-titer volatile fatty acids and generate organic solvents from renewable carbon sources including biowaste and CO2 . In this study, four phosphonium ionic liquids were tested for single-stage extraction of acetic acid from a dilute stream and esterification to ethyl acetate with added ethanol and heat. The esterification proceeded with a maximum conversion of 85.9±1.3 % after 30 min at 75 °C at a 1:1 stoichiometric ratio of reactants. Extraction and esterification can be tailored using mixed-anion ionic liquids; this is demonstrated herein using a common trihexyl(tetradecyl)phosphonium cation and a mixed chloride and bis(trifluoromethylsulfonyl)imide anion ionic liquid. As a further proof-of-concept, ethyl acetate was generated from an ionic liquid-driven esterification of an acetic acid extractant generated using CO2 as the only carbon source by microbial electrosynthesis.

A logical data representation framework for electricity-driven bioproduction processes.

Microbial electrosynthesis (MES) is a process that uses electricity as an energy source for driving the production of chemicals and fuels using microorganisms and CO2 or organics as carbon sources. The development of this highly interdisciplinary technology on the interface between biotechnology and electrochemistry requires knowledge and expertise in a variety of scientific and technical areas. The rational development and commercialization of MES can be achieved at a faster pace if the research data and findings are reported in appropriate and uniformly accepted ways. Here we provide a framework for reporting on MES research and propose several pivotal performance indicators to describe these processes. Linked to this study is an online tool to perform necessary calculations and identify data gaps. A key consideration is the calculation of effective energy expenditure per unit product in a manner enabling cross comparison of studies irrespective of reactor design. We anticipate that the information provided here on different aspects of MES ranging from reactor and process parameters to chemical, electrochemical, and microbial functionality indicators will assist researchers in data presentation and ease data interpretation. Furthermore, a discussion on secondary MES aspects such as downstream processing, process economics and life cycle analysis is included.

Electrochemically and bioelectrochemically induced ammonium recovery.

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ammonium from an ammonium-rich stream is demonstrated using an electrochemical and a bioelectrochemical system. Both systems are controlled by a potentiostat to either fix the current (for the electrochemical cell) or fix the potential of the working electrode (for the bioelectrochemical cell). In the bioelectrochemical cell, electroactive bacteria catalyze the anodic reaction, whereas in the electrochemical cell the potentiostat applies a higher voltage to produce a current. The current and consequent restoration of the charge balance across the cell allow the transport of cations, such as ammonium, across a cation exchange membrane from the anolyte to the catholyte. The high pH of the catholyte leads to formation of ammonia, which can be stripped from the medium and captured in an acid solution, thus enabling the recovery of a valuable nutrient. The flux of ammonium across the membrane is characterized at different anolyte ammonium concentrations and currents for both the abiotic and biotic reactor systems. Both systems are compared based on current and removal efficiencies for ammonium, as well as the energy input required to drive ammonium transfer across the cation exchange membrane. Finally, a comparative analysis considering key aspects such as reliability, electrode cost, and rate is made. This video article and protocol provide the necessary information to conduct electrochemical and bioelectrochemical ammonia recovery experiments. The reactor setup for the two cases is explained, as well as the reactor operation. We elaborate on data analysis for both reactor types and on the advantages and disadvantages of bioelectrochemical and electrochemical systems.

Inoculum selection is crucial to ensure operational stability in anaerobic digestion.

Anaerobic digestion is considered a key technology for the future bio-based economy. The microbial consortium carrying out the anaerobic digestion process is quite complex, and its exact role in terms of "elasticity", i.e., the ability to rapidly adapt to changing conditions, is still unknown. In this study, the role of the initial microbial community in terms of operational stability and stress tolerance was evaluated during a 175-day experiment. Five different inocula from stable industrial anaerobic digesters were fed a mixture of waste activated sludge and glycerol. Increasing ammonium pulses were applied to evaluate stability and stress tolerance. A different response in terms of start-up and ammonium tolerance was observed among the different inocula. Methanosaetaceae were the dominant acetoclastic methanogens, yet, Methanosarcinaceae increased in abundance at elevated ammonium concentrations. A shift from a Firmicutes to a Proteobacteria dominated bacterial community was observed in failing digesters. Methane production was strongly positively correlated with Methanosaetaceae, but also with Bacteria related to Anaerolinaceae, Clostridiales, and Alphaproteobacteria. Volatile fatty acids were strongly positively correlated with Betaproteobacteria and Bacteroidetes, yet ammonium concentration only with Bacteroidetes. Overall, these results indicate the importance of inoculum selection to ensure stable operation and stress tolerance in anaerobic digestion.

Electrolytic membrane extraction enables production of fine chemicals from biorefinery sidestreams.

Short-chain carboxylates such as acetate are easily produced through mixed culture fermentation of many biological waste streams, although routinely digested to biogas and combusted rather than harvested. We developed a pipeline to extract and upgrade short-chain carboxylates to esters via membrane electrolysis and biphasic esterification. Carboxylate-rich broths are electrolyzed in a cathodic chamber from which anions flux across an anion exchange membrane into an anodic chamber, resulting in a clean acid concentrate with neither solids nor biomass. Next, the aqueous carboxylic acid concentrate reacts with added alcohol in a water-excluding phase to generate volatile esters. In a batch extraction, 96 ± 1.6% of the total acetate was extracted in 48 h from biorefinery thin stillage (5 g L(-1) acetate) at 379 g m(-2) d(-1) (36% Coulombic efficiency). With continuously regenerated thin stillage, the anolyte was concentrated to 14 g/L acetic acid, and converted at 2.64 g (acetate) L(-1) h(-1) in the first hour to ethyl acetate by the addition of excess ethanol and heating to 70 °C, with a final total conversion of 58 ± 3%. This processing pipeline enables direct production of fine chemicals following undefined mixed culture fermentation, embedding carbon in industrial chemicals rather than returning them to the atmosphere as carbon dioxide.

Biomass retention on electrodes rather than electrical current enhances stability in anaerobic digestion.

Anaerobic digestion (AD) is a well-established technology for energy recovery from organic waste streams. Several studies noted that inserting a bioelectrochemical system (BES) inside an anaerobic digester can increase biogas output, however the mechanism behind this was not explored and primary controls were not executed. Here, we evaluated whether a BES could stabilize AD of molasses. Lab-scale digesters were operated in the presence or absence of electrodes, in open (no applied potential) and closed circuit conditions. In the control reactors without electrodes methane production decreased to 50% of the initial rate, while it remained stable in the reactors with electrodes, indicating a stabilizing effect. After 91 days of operation, the now colonized electrodes were introduced in the failing AD reactors to evaluate their remediating capacity. This resulted in an immediate increase in CH4 production and VFA removal. Although a current was generated in the BES operated in closed circuit, no direct effect of applied potential nor current was observed. A high abundance of Methanosaeta was detected on the electrodes, however irrespective of the applied cell potential. This study demonstrated that, in addition to other studies reporting only an increase in methane production, a BES can also remediate AD systems that exhibited process failure. However, the lack of difference between current driven and open circuit systems indicates that the key impact is through biomass retention, rather than electrochemical interaction with the electrodes.