Effect of n-Caproate Concentration on Chain Elongation and Competing Processes.

Chain elongation is an open-culture fermentation process that facilitates conversion of organic residues with an additional electron donor, such as ethanol, into valuable n-caproate. Open-culture processes are catalyzed by an undefined consortium of microorganisms which typically also bring undesired (competing) processes. Inhibition of competing processes, such as syntrophic ethanol oxidation, will lead to a more selective n-caproate production process. In this study, we investigated the effect of n-caproate concentration on the specific activity of chain elongation and competing processes using batch inhibition assays. With "synthetic medium sludge" (originally operating at 3.4 g/L n-caproate), syntrophic ethanol oxidation was proportionally inhibited by n-caproate until 45% inhibition at 20 g/L n-caproate. Hydrogenotrophic methanogenesis was for 58% inhibited at 20 g/L n-caproate. Chain elongation of volatile fatty acids (volatile fatty acid upgrading; the desired process), was completely inhibited at 20 g/L n-caproate with all tested sludge types. "Adapted sludge" (operating at 23.2 g/L n-caproate) showed a 10 times higher volatile fatty acid upgrading activity at 15 g/L n-caproate compared to "nonadapted sludge" (operating at 7.1 g/L n-caproate). This shows that open cultures do adapt to perform chain elongation at high n-caproate concentrations which likely inhibits syntrophic ethanol oxidation through hydrogenotrophic methanogenesis. As such, we provide supporting evidence that the formation of n-caproate inhibits syntrophic ethanol oxidation which leads to a more selective medium chain fatty acid production process.

Development of an Effective Chain Elongation Process From Acidified Food Waste and Ethanol Into n-Caproate.

Introduction: Medium chain fatty acids (MCFAs), such as n-caproate, are potential valuable platform chemicals. MCFAs can be produced from low-grade organic residues by anaerobic reactor microbiomes through two subsequent biological processes: hydrolysis combined with acidogenesis and chain elongation. Continuous chain elongation with organic residues becomes effective when the targeted MCFA(s) are produced at high concentrations and rates, while excessive ethanol oxidation and base consumption are limited. The objective of this study was to develop an effective continuous chain elongation process with hydrolyzed and acidified food waste and additional ethanol. Results: We fed acidified food waste (AFW) and ethanol to an anaerobic reactor while operating the reactor at long (4 d) and at short (1 d) hydraulic retention time (HRT). At long HRT, n-caproate was continuously produced (5.5 g/L/d) at an average concentration of 23.4 g/L. The highest n-caproate concentration was 25.7 g/L which is the highest reported n-caproate concentration in a chain elongation process to date. Compared to short HRT (7.1 g/L n-caproate at 5.6 g/L/d), long HRT resulted in 6.2 times less excessive ethanol oxidation. This led to a two times lower ethanol consumption and a two times lower base consumption per produced MCFA at long HRT compared to short HRT. Conclusions: Chain elongation from AFW and ethanol is more effective at long HRT than at short HRT not only because it results in a higher concentration of MCFAs but also because it leads to a more efficient use of ethanol and base. The HRT did not influence the n-caproate production rate. The obtained n-caproate concentration is more than twice as high as the maximum solubility of n-caproic acid in water which is beneficial for its separation from the fermentation broth. This study does not only set the record on the highest n-caproate concentration observed in a chain elongation process to date, it notably demonstrates that such high concentrations can be obtained from AFW under practical circumstances in a continuous process.

Controlling Ethanol Use in Chain Elongation by CO2 Loading Rate.

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO2 loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO2 to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO2 loading rate (2.5 LCO2·L-1·d-1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L-1·d-1). A low CO2 loading rate (0.5 LCO2·L-1·d-1) reduced EEO (16%) and caproate production (2.9 g·L-1·d-1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L-1·d-1) at CO2 loading rates higher than or equal to 1 LCO2·L-1·d-1. CO2 was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO2 loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO2 loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

Novel method for enumeration of viable Lactobacillus plantarum WCFS1 cells after single-droplet drying.

Survival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale.