Catherine M. Spirito

Chain elongation with reactor microbiomes: upgrading dilute ethanol to medium-chain carboxylates

Ethanol distillation in the biofuel industry is energetically expensive because ethanol is completely miscible in water. Upgrading ethanol into a hydrophobic chemical that is easier to separate would circumvent current fossil-fuel consumption for distillation. Here, we shaped a reactor microbiome to sequentially elongate carboxylic acids with 2-carbon units from dilute ethanol in yeast-fermentation beer. Our continuous bioprocess produced n-caproic acid, a 6-carbon-chain carboxylic acid that is more valuable than ethanol. No antimicrobials to inhibit methanogens were necessary. In-line product extraction achieved an n-caproic acid production rate exceeding 2 grams per liter of reactor volume per day, which is comparable to established bioenergy systems with microbiomes. Incorporation of other organics found in beer increased the mass of carbon in n-caproic acid by 10% compared to ethanol.

Chain elongation in anaerobic reactor microbiomes to recover resources from waste

Different microbial pathways can elongate the carbon chains of molecules in open cultures of microbial populations (i.e. reactor microbiomes) under anaerobic conditions. Here, we discuss three such pathways: 1. homoacetogenesis to combine two carbon dioxide molecules into acetate; 2. succinate formation to elongate glycerol with one carbon from carbon dioxide; and 3. reverse β oxidation to elongate short-chain carboxylates with two carbons into medium-chain carboxylates, leading to more energy-dense and insoluble products (e.g. easier to separate from solution). The ability to use reactor microbiomes to treat complex substrates can simultaneously address two pressing issues: 1. providing proper waste management; and 2. producing renewable chemicals and fuels.

Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals

Chain elongation into medium-chain carboxylates, such as n-caproate and n-caprylate, with ethanol as an electron donor and with open cultures of microbial consortia (i.e., reactor microbiomes) under anaerobic conditions is being developed as a biotechnological production platform. The goal is to use the high thermodynamic efficiency of anaerobic fermentation to convert organic biomass or organic wastes into valuable biochemicals that can be extracted. Several liter-scale studies have been completed and a first pilot-plant study is underway. However, the underlying microbial pathways are not always well understood. In addition, an interdisciplinary approach with knowledge from fields ranging from microbiology and chemical separations to biochemistry and environmental engineering is required. To bring together research from different fields, we reviewed the literature starting with the microbiology and ending with the bioprocess engineering studies that already have been performed. Because understanding the microbial pathways is so important to predict and steer performance, we delved into a stoichiometric and thermodynamic model that sheds light on the effect of substrate ratios and environmental conditions on product formation. Finally, we ended with an outlook.

Long-Term n-Caproic Acid Production from Yeast-Fermentation Beer in an Anaerobic Bioreactor with Continuous Product Extraction

Multifunctional reactor microbiomes can elongate short-chain carboxylic acids (SCCAs) to medium-chain carboxylic acids (MCCAs), such as n-caproic acid. However, it is unclear whether this microbiome biotechnology platform is stable enough during long operating periods to consistently produce MCCAs. During a period of 550 days, we improved the operating conditions of an anaerobic bioreactor for the conversion of complex yeast-fermentation beer from the corn kernel-to-ethanol industry into primarily n-caproic acid. We incorporated and improved in-line, membrane liquid-liquid extraction to prevent inhibition due to undissociated MCCAs at a pH of 5.5 and circumvented the addition of methanogenic inhibitors. The microbiome accomplished several functions, including hydrolysis and acidogenesis of complex organic compounds and sugars into SCCAs, subsequent chain elongation with undistilled ethanol in beer, and hydrogenotrophic methanogenesis. The methane yield was 2.40 ± 0.52% based on COD and was limited by the availability of carbon dioxide. We achieved an average n-caproate production rate of 3.38 ± 0.42 g L(-1) d(-1) (7.52 ± 0.94 g COD L(-1) d(-1)) with an n-caproate yield of 70.3 ± 8.81% and an n-caproate/ethanol ratio of 1.19 ± 0.15 based on COD for a period of ∼55 days. The maximum production rate was achieved by increasing the organic loading rates in tandem with elevating the capacity of the extraction system and a change in the complex feedstock batch.

Development of a highly specific and productive process for n-caproic acid production: applying lessons from methanogenic microbiomes

High productivity and specificity in anaerobic digesters arise because complex microbiomes organize into a metabolic cascade to maximize energy recovery and to utilize the advantage that the gaseous end product methane freely bubbles out of the system. These lessons were applied to ascertain whether a reactor microbiome could be shaped to produce a different end product. The liquid product n-caproic acid was chosen, which is a 6-carbon-chain carboxylic acid that is valuable and that has a relatively low maximum solubility concentration for product recovery. Acetoclastic methanogenesis was inhibited by pH control and a route was provided for n-caproic acid extraction by implementing selective, in-line recovery. Next, ethanol was supplemented to promote chain elongation, which is a pathway in which short-chain carboxylic acids are elongated sequentially into medium-chain carboxylic acids with two-carbon units derived from ethanol. The reactor microbiome developed accordingly with the terminal process catalyzed by chain-elongating bacteria. As a result, n-caproic acid production rates increased to levels comparable to anaerobic digestion systems for solid waste treatment.

High n-caprylate productivities and specificities from dilute ethanol and acetate: chain elongation with microbiomes to upgrade products from syngas fermentation

Past studies on chain elongation by reactor microbiomes have shown promising productivities and specificities of n-caproate, which is a six-carbon (C6) medium-chain carboxylate (MCC). The production of small quantities of n-caprylate (C8) was also reported, but merely as a by-product of n-caproate production. Since n-caprylate is considerably more valuable than n-caproate by weight, the question was raised whether n-caprylate could be produced at a high specificity (product vs. all other carboxylate products). Here, we optimized the operating conditions of an anaerobic upflow bioreactor during a period of 186 days to accomplish this goal. We achieved an n-caprylate productivity of 19.4 g chemical oxygen demand (COD) L−1 d−1 (0.33 g L−1 h−1), which is the highest ever reported. The product ratio of n-caprylate to n-caproate was 11 g COD per g COD, which was also greater than past studies. This ratio was even 25 g COD per g COD during an earlier operating period with a lower productivity, resulting in an n-caprylate specificity of 96% (based on COD). We accomplished this high n-caprylate productivity and specificity by: (1) feeding a substrate with a high substrate ratio of ethanol to acetate; (2) extracting the product from the bioreactor broth; and (3) acclimating an efficient chain-elongating microbiome. Because syngas-fermentation effluent contains a high ratio of ethanol to acetate, we propose coupling syngas fermentation with chain elongation to produce n-caprylate and to increase product value.

Comparing the inhibitory thresholds of dairy manure co-digesters after prolonged acclimation periods: Part 2--correlations between microbiomes and environment.

Here, we studied the microbiome succession and time-scale variability of four mesophilic anaerobic reactors in a co-digestion study with the objective to find links between changing environmental conditions and the microbiome composition. The changing environmental conditions were ensured by gradual increases in loading rates and mixing ratios of three co-substrates with a constant manure-feeding scheme during an operating period longer than 900 days. Each co-substrate (i.e., alkaline hydrolysate, food waste, and glycerol) was co-digested separately. High throughput 16S rRNA gene sequencing was used to examine the microbiome succession. The alkaline hydrolysate reactor microbiome shifted and adapted to high concentrations of free ammonia, total volatile fatty acids, and potassium to maintain its function. The addition of food waste and glycerol as co-substrates also led to microbiome changes, but to a lesser extent, especially in the case of the glycerol reactor microbiome. The divergence of the food waste reactor microbiome was primarily linked to increasing free ammonia levels in the reactor; though, these levels remained below previously reported inhibitory levels for acclimated biomass. The glycerol reactor microbiome succession included an increase in Syntrophomonadaceae family members, which have previously been linked to long-chain fatty acid degradation. The glycerol reactor exhibited rapid failure and limited adaptation at the end of the study.

Continuously-stirred anaerobic digester to convert organic wastes into biogas: system setup and basic operation.

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale. Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs). This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation.

Higher Substrate Ratios of Ethanol to Acetate Steered Chain Elongation Toward n-Caprylate in a Bioreactor with Product Extraction

Syngas fermentation to ethanol and acetate has recently been coupled to microbial chain elongation to produce medium-chain carboxylates, including n-caproate and n-caprylate. These medium-chain carboxylates are relatively hydrophobic, and thus easier to extract from solution than miscible ethanol. Here, we examined the effect of 11 different ethanol-to-acetate substrate ratios (ranging from 1.8 to 275 g COD g COD-1 [1.2 to 183 mol mol-1]) on directing chain elongation toward n-caprylate in a 0.7-L upflow anaerobic filter with product extraction. During an eight-month operating period, we monitored the performance and characterized the microbiome composition of this chain-elongating bioreactor. We also developed a thermodynamic model to predict the favorability of n-caprylate production at different substrate ratios. As predicted by our model, higher ethanol-to-acetate substrate ratios fed to our bioreactor led to higher specificities for n-caprylate production. We observed that feeding primarily ethanol to the bioreactor (i.e., ethanol-to-acetate substrate ratio of 275 g COD g COD-1) resulted in the highest specificity for n-caprylate, but the n-caprylate production rate decreased at this high ratio, resulting in lower conversion efficiencies. Thus, care should be taken not to overload the system with primarily ethanol as the substrate and to lower the organic loading rate.

Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

ABSTRACT The antibiotic monensin is fed to dairy cows to increase milk production efficiency. A fraction of this monensin is excreted into the cow manure. Previous studies have found that cow manure containing monensin can negatively impact the performance of anaerobic digesters, especially upon first introduction. Few studies have examined whether the anaerobic digester microbiome can adapt to monensin during the operating time. Here, we conducted a long-term time series study of four lab-scale anaerobic digesters fed with cow manure. We examined changes in both the microbiome composition and function of the anaerobic digesters when subjected to the dairy antibiotic monensin. In our digesters, monensin was not rapidly degraded under anaerobic conditions. The two anaerobic digesters that were subjected to manure from monensin feed-dosed cows exhibited relatively small changes in microbiome composition and function due to relatively low monensin concentrations. At higher concentrations of monensin, which we dosed directly to control manure (from dairy cows without monensin), we observed major changes in the microbiome composition and function of two anaerobic digesters. A rapid introduction of monensin to one of these anaerobic digesters led to the impairment of methane production. Conversely, more gradual additions of the same concentrations of monensin to the other anaerobic digester led to the adaptation of the anaerobic digester microbiomes to the relatively high monensin concentrations. A member of the candidate OP11 (Microgenomates) phylum arose in this anaerobic digester and appeared to be redundant with certain Bacteroidetes phylum members, which previously were dominating. IMPORTANCE Monensin is a common antibiotic given to dairy cows in the United States and is partly excreted with dairy manure. An improved understanding of how monensin affects the anaerobic digester microbiome composition and function is important to prevent process failure for farm-based anaerobic digesters. This time series study demonstrates how anaerobic digester microbiomes are inert to low monensin concentrations and can adapt to relatively high monensin concentrations by redundancy in an already existing population. Therefore, our work provides further insight into the importance of microbiome redundancy in maintaining the stability of anaerobic digesters.