Joseph G. Usack

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.

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.

Comparing the inhibitory thresholds of dairy manure co-digesters after prolonged acclimation periods: Part 1 – Performance and operating limits

Co-digestion has been used to improve biogas yields and the long-term stability of anaerobic digesters compared to mono-digestion; however, less is known about the ultimate inhibition from co-substrates at their maximum loading rates and mixing ratios because these limits cannot be practically tested by existing facilities. Here, we performed a controlled experiment with long operating periods to ensure sufficient acclimation with the goal to observe ultimate inhibition and the full benefit that can be gained from co-digestion. The three substrates: 1) food waste (FW); 2) alkaline hydrolysate (AH); and 3) crude glycerol (GY) were individually co-digested with dairy manure (MN) for more than 900 days using continuously stirred anaerobic reactors at mesophilic temperatures. Food waste caused no reduction in performance or stability when co-digested with manure up to a total organic loading rate (OLR) of 3.9 g volatile solids (VS)·L(-1)·Day(-1) (MN:FW = 51:49; VS basis), resulting in a specific methane yield (SMY) of 297 ± 3 mL CH4·g VS(-1) for the combined wastes. Alkaline hydrolysate was co-digested with manure up to a total OLR of 2.7 g VS·L(-1)·Day(-1) (MN:AH = 75:25) with a corresponding SMY of 299 ± 6 mL CH4·g VS(-1). However, the free ammonia concentration reached levels previously reported as inhibitory, and may have led to the observed accumulation of volatile fatty acids at higher loading rates. Crude glycerol co-digestion resulted in an optimum SMY of 549 ± 25 mL CH4·g VS(-1) at a total OLR of 3.2 g VS·L(-1)·Day(-1) (MN:GY = 62:38). Stable digestion beyond this level was prohibited by an accumulation of long-chain fatty acids and foaming. These results can be used to implement effective co-digestion strategies. Co-substrates that possess similar inhibiting characteristics should be monitored to prevent severe instability at high loading rates and mixing ratios.

Coupling hydrothermal liquefaction and anaerobic digestion for energy valorization from model biomass feedstocks

Hydrothermal liquefaction converts food waste into oil and a carbon-rich hydrothermal aqueous phase. The hydrothermal aqueous phase may be converted to biomethane via anaerobic digestion. Here, the feasibility of coupling hydrothermal liquefaction and anaerobic digestion for the conversion of food waste into energy products was examined. A mixture of polysaccharides, proteins, and lipids, representing food waste, underwent hydrothermal processing at temperatures ranging from 200 to 350°C. The anaerobic biodegradability of the hydrothermal aqueous phase was examined through conducting biochemical methane potential assays. The results demonstrate that the anaerobic biodegradability of the hydrothermal aqueous phase was lower when the temperature of hydrothermal processing increased. The chemical composition of the hydrothermal aqueous phase affected the anaerobic biodegradability. However, no inhibition of biodegradation was observed for most samples. Combining hydrothermal and anaerobic digestion may, therefore, yield a higher energetic return by converting the feedstock into oil and biomethane.

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.

Production and physiological responses of heat-stressed lactating dairy cattle to conductive cooling.

The objective of this research was to test the effectiveness of conductive cooling in alleviating heat stress of lactating dairy cows. A conductive cooling system was built with waterbeds (Dual Chamber Cow Waterbeds, Advanced Comfort Technology Inc., Reedsburg, WI) modified to circulate chilled water. The experiment lasted 7 wk. Eight first-lactation Holstein cows producing 34.4±3.7kg/d of milk at 166±28 d in milk were used in the study. Milk yield, dry matter intake (DMI), and rectal temperature were recorded twice daily, and respiration rate was recorded 5 times per day. During wk 1, the cows were not exposed to experimental heat stress or conductive cooling. For the remaining 6 wk, the cows were exposed to heat stress from 0900 to 1700h each day. During these 6 wk, 4 of the 8 cows were cooled with conductive cooling (experimental cows), and the other 4 were not cooled (control cows). The study consisted of 2 thermal environment exposures (temperature-humidity index mean ± standard deviation of 80.7±0.9 and 79.0±1.0) and 2 cooling water temperatures (circulating water through the water mattresses at temperatures of 4.5°C and 10°C). Thus, a total of 4 conductive cooling treatments were tested, with each treatment lasting 1 wk. During wk 6, the experimental and control cows were switched and the temperature-humidity index of 79.0±1.0 with 4.5°C cooling water treatment was repeated. During wk 7, waterbeds were placed directly on concrete stalls without actively cooling the water. Least squares means and P-values for the different treatments were calculated with multivariate mixed models. Conductively cooling the cows with 4.5°C water decreased rectal temperature by 1.0°C, decreased respiration rate by 18 breaths/min, increased milk yield by 5%, and increased DMI by 14% compared with the controls. When the results from the 2 cooling water temperatures (4.5°C and 10°C circulating water) were compared, we found that the rectal temperature from 4.5°C cooling water was 0.3°C lower than the rectal temperature with 10°C cooling water, but the other measurements (respiration rate, milk production, and DMI) did not show a statistically significant difference between the cooling water temperatures. Placing waterbeds on concrete stalls without additional cooling did not have a measurable effect in alleviating the heat stress of the cows.

Improved design of anaerobic digesters for household biogas production in indonesia: one cow, one digester, and one hour of cooking per day.

A government-sponsored initiative in Indonesia to design and implement low-cost anaerobic digestion systems resulted in 21 full-scale systems with the aim to satisfy the cooking fuel demands of rural households owning at least one cow. The full-scale design consisted of a 0.3 m diameter PVC pipe, which was operated as a conventional plug-flow system. The system generated enough methane to power a cooking stove for ∼1 h. However, eventual clogging from solids accumulation inside the bioreactor proved to be a major drawback. Here, we improved the digester configuration to remedy clogging while maintaining system performance. Controlled experiments were performed using four 9-L laboratory-scale digesters operated at a temperature of 27 ± 1°C, a volatile solids loading rate of 2.0 g VS·L−1 ·day−1, and a 21-day hydraulic retention time. Two of the digesters were replicates of the original design (control digesters), while the other two digesters included internal mixing or effluent recycle (experimental digesters). The performance of each digester was compared based on methane yields, VS removal efficiencies, and steady-state solids concentrations during an operating period of 311 days. Statistical analyses revealed that internal mixing and effluent recycling resulted in reduced solids accumulation compared to the controls without diminishing methane yields or solids removal efficiencies.

Integrating electrochemical, biological, physical, and thermochemical process units to expand the applicability of anaerobic digestion

Anaerobic digestion (AD) is a mature biotechnology-production platform with millions of installations at homes, farms, and industrial/municipal settings. Large-scale industrial, agricultural, and municipal waste-treatment systems may observe novel integration with electrochemical, biological, physical, and thermochemical process units to make AD more attractive. Without governmental subsidies, AD has often only a relatively low economic return or none at all. Diversification of products besides methane in biogas may help to change this. Here, several sections discuss different process units to: 1) upgrade biogas into biomethane; 2) convert carbon dioxide in biogas to more biomethane; 3) generate cooling power from process heat; 4) produce bio-crude oil (bio-oil) from organic matter; and 5) produce a liquid biochemical product from organic matter. This is not meant to be an exhaustive list, but rather a selection of particularly promising process units from a technological view, which are already integrated with AD or close to full-scale integration.