Biology subjects

Usack, J. G.

Publications and source records attributed to Usack, J. G..

7 recordsLinked to original sources

bioRxiv2025

Micro-aeration-enhanced Anaerobic Digestion for the Stabilization of Coffee-Processing Wastewater

Coffee processing wastewater (CPW), a byproduct of agro-industrial operations, contains high organic loads alongside recalcitrant and potentially inhibitory compounds such as caffeine and tannins. This study evaluated the performance of micro-aeration-enhanced anaerobic digestion (MA-AD) for the treatment and valorization of CPW to promote a more sustainable approach to coffee production. Oxygen was intermittently introduced via oxidation-reduction potential-controlled dosing, allowing for comparative assessment across anaerobic and micro-aerobic redox regimes. While both conventional anaerobic digestion (AD) and MA-AD achieved comparable reductions in total and volatile solids (>48% and >60%, respectively) and total and soluble chemical oxygen demand (>66% and >86%, respectively), MA-AD exhibited significantly higher total suspended solids concentrations and turbidity in later phases, likely due to gas sparging-induced floc disruption and particulate release. pH profiles indicated a shift toward increased acidification under MA-AD, without compromising process stability, with both reactors stabilizing between pH 6.8-7.1. Caffeine degradation was accelerated under MA-AD in the first dosing phase (>85% removal in 28 h), though long-term degradation efficiency converged with the control. Methane production was consistently lower in MA-AD (up to 43% reduction), attributed to the oxygen sensitivity of methanogens and possible substrate competition. These results underscore the importance of oxygen dose regulation, redox control, and microbial adaptation in optimizing MA-AD performance. The findings support MA-AD as a promising strategy for enhancing hydrolysis and partial removal of recalcitrant compounds in CPW. However, further refinement is required to sustain biogas quality and yield at scale.

bioengineering↗

bioRxiv2025

Design and validation of a new flat-sheet membrane bioreactor system for bioprocessing research requiring very low gas fluxes

Precise and consistent gas dosing at trace levels remains a significant challenge in bioprocessing and biotechnology research. This study introduces a novel flat-sheet membrane bioreactor for controlled gas delivery without bubble formation. The system relies on gas diffusion mediated by interchangeable membrane cassettes and incorporates mechanical stirring near the membrane surface to promote gas dispersal and mitigate biofouling spanning long operating periods. Comprehensive characterization of mass transfer properties using oxygen gas as a test case revealed that the volumetric mass transfer coefficient (KLa) remained stable under varying operational conditions, including gas partial pressure, back pressure, and total gas flow rate. The maximum oxygen flux under specific operational conditions flexibly ranged from 15.9E-03 {+/-} 6.3E-03 mol{middle dot}min-1 to 1.08 {+/-} 0.17 mol{middle dot}min-1 for various porous membranes and from 23.3E-03 {+/-} 3.5E-03 mol{middle dot}min-1 to 0.161 {+/-} 0.044 mol{middle dot}min-1 for the non-porous membranes, indicating the bioreactor can serve as an experimental platform for a variety of applications. A biological validation study using starch-containing wastewater demonstrated the feasibility of continuous microaerobic oxygen dosing, achieving reproducible performance and maintaining stable operation over 320 days. These findings highlight the potential of the developed system as a reliable experimental platform for investigating bioprocesses dependent on trace gas supply. Future improvements should focus on scaling up the system, addressing porous membrane wetting, and expanding its applicability to other gases and bioprocessing applications.

bioengineering↗

bioRxiv2024

Toxicity and Biodegradation of Two Different Hydrothermal Liquefaction Process Waters to Anaerobic Digestion and the Effect of Microaeration

Hydrothermal liquefaction (HTL) can convert a considerable portion of the carbon in complex feedstocks into renewable bio-oil, but it also generates a liquid byproduct (i.e., HTL process water) that retains 15 - 55% of the carbon from the HTL feedstock. Feeding HTL process water to anaerobic digestion (AD) is a promising approach for maximizing resource recovery, enabling the conversion of the retained carbon into biogas. However, various toxic and poorly biodegradable compounds in HTL process water make its treatment with AD challenging. Presently, the underlying mechanisms remain often unclear. We investigated the impact of HTL process water from two different feedstocks - a food-waste proxy (i.e., dog food, rich in proteins) and wheat straw (rich in lignocellulose) - on the different trophic groups in the food web of AD. We found that methanogens rather than acidogens were inhibited by HTL process water. Comparative toxicity and biodegradability analyses showed that wheat-straw process water had a higher biodegradability regardless of its higher toxicity to acetoclastic methanogens than food-waste process water, due to its higher content in toxic but easily degradable aromatic compounds. Microaeration enhanced the biodegradation and methane yields of food-waste process water, particularly under anoxic conditions. However, microaeration was ineffective for wheat-straw process water. These findings highlight the importance of feedstock-specific strategies to optimize AD for biogas production from HTL process water.

bioengineering↗

bioRxiv2024

Lactate production from lactose-rich wastewater: A comparative study on reactor configurations to maximize conversion rates and efficiencies

About 90% of global lactate production is derived from bacterial fermentation of sugars via pure cultures of homofermentative bacteria in batch mode. Acid whey, which is a wastewater from the yogurt industry with lactose and galactose as the main sugars, can be used as an alternative substrate for the commercial production of lactate. Operating open cultures of microbial consortia (i.e., reactor microbiomes) reduces the costs of lactate production by circumventing sterilization, while continuous operation achieves higher productivity at shorter production times. Homofermentation can be achieved by maintaining acidic and thermophilic conditions, while product formation in continuous systems can be increased with biomass retention strategies. To find the best reactor configuration for lactate production from acid whey, we operated three different reactor configurations: (1) an upflow anaerobic sludge blanket (UASB) reactor; (2) an anaerobic filter reactor (AFR); and (3) an anaerobic continuously stirred tank reactor (CSTR) with a hollow-fiber membrane module. We operated at different hydraulic retention times (HRTs) to find the optimum parameters to maximize the lactose and galactose-into-lactate (LG-into-LA) conversion efficiency. We did not use an inoculum but enriched the endogenous D-lactate-producing Lactobacillus spp. that later dominated the reactor microbiomes (> 90% relative abundance). Undissociated lactic acid concentrations of more than 60 mmol C L-1 inhibited the microbiomes. We alleviated the inhibition effect by shortening the HRT to 0.6 days and using diluted acid-whey substrate (1.67-fold dilution) to achieve almost complete conversion of the acid-whey sugars to lactate. At the 0.6-day HRT commencement, the AFR and CSTR performed better than the UASB reactor due to their better cell retention abilities. During the period between Day 365-384, we experienced an error in the pH control of the CSTR system during which the pH value dropped to 4.3. After this pH-error period, the LG-into-LA conversion efficiency for the CSTR considerably improved and surpassed the AFR. We achieved the highest lactate conversion rate of 1256 {+/-} 46.3 mmol C L-1 d-1 (1.57 {+/-} 0.06 g L-1 h-1) at a LG-into-LA conversion efficiency of 82.2 {+/-} 3.4% (in mmol C), with a yield of 0.85 {+/-} 0.02 mmol C mmol C-1 (product per consumed substrate) for the CSTR.

bioengineering↗

bioRxiv2024

Toward industrial C8 production: Oxygen intrusion drives renewable n caprylate production from ethanol and acetate via intermediate metabolite production

Previous bioreactor studies achieved high volumetric n-caprylate (i.e., n-octanoate) production rates and selectivities from ethanol and acetate with chain-elongating microbiomes. However, the metabolic pathways from the substrates to n-caprylate synthesis were unclear. We operated two n-caprylate-producing upflow bioreactors with a synthetic medium to study the underlying metabolic pathways. The operating period exceeded 2.5 years, with a peak volumetric n-caprylate production rate of 190 {+/-} 8.4 mmol C L-1 d-1 (0.14 g L-1 h-1). We identified oxygen availability as a critical performance parameter, facilitating intermediate metabolite production from ethanol. Bottle experiments in the presence and absence of oxygen with 13C-labeled ethanol suggest acetyl-coenzyme A-based derived production of n-butyrate (i.e., n-butanoate), n-caproate (i.e., n-hexanoate), and n-caprylate. Here, we postulate a trophic hierarchy within the bioreactor microbiomes based on metagenomics, metaproteomics, and metabolomics data, as well as experiments with a Clostridium kluyveri isolate. First, the aerobic bacterium Pseudoclavibacter caeni and the facultative anaerobic fungus Cyberlindnera jadinii converted part of the ethanol pool into the intermediate metabolites succinate, lactate, and pyroglutamate. Second, the strict anaerobic C. kluyveri elongated acetate with the residual ethanol to n-butyrate. Third, Caproicibacter fermentans and Oscillibacter valericigenes elongated n-butyrate with the intermediate metabolites to n-caproate and then to n-caprylate. Among the carbon chain-elongating pathways of carboxylates, the tricarboxylic acid cycle and the reverse {beta}-oxidation pathways showed a positive correlation with n-caprylate production. The results of this study inspire the realization of a chain-elongating production platform with separately controlled aerobic and anaerobic stages to produce n-caprylate renewably as an attractive chemical from ethanol and acetate as substrates. Broader contextNext to renewable electric energy, carbon-based chemicals have to be produced sustainably and independently from fossil sources. To meet this goal, we must expand the portfolio of bio-based conversion technologies on an industrial scale to cover as many target chemicals as possible. We explore the bioprocess of chain elongation to provide medium-chain carboxylates that can function as future platform chemicals in the circular economy. The most valuable medium-chain carboxylate produced with chain elongation is n-caprylate (i.e., n-octanoate). This molecule with eight carbon atoms in a row (C8) is challenging to produce renewably for the chemical industry. Previous reports elucidated that elevated ethanol-to-acetate ratios, which are found in syngas-fermentation effluent, stimulated n-caprylate production. Until now, studies have suggested that chain elongation from high concentrations of ethanol and acetate is a fully anaerobic process. We refine this view by showing a trophic hierarchy of aerobic and anaerobic microbes capable of facilitating this process. Appropriate oxygen supplementation enables the synthesis of succinate, lactate, and pyroglutamate that permit high-rate chain elongation to n-caprylate under anaerobic conditions. Given these results, future research should focus on the segregated study of aerobic and anaerobic microbes to further enhance the process performance to produce n-caprylate renewably at an industrial scale.

bioengineering↗

bioRxiv2022

Near-neutral pH increased n-caprylate production in a microbiome with product inhibition of methanogenesis

The pH is a critical parameter in chain-elongating bioreactors, affecting: (1) the concentration of inhibitory undissociated carboxylic acids, which in turn affects the efficiency of product extraction; (2) the thermodynamics; and (3) the kinetics. Here, we examined the effect of five different pH levels (5.5 to 7.0) on n-caprylate (C8) production using an anaerobic sequencing batch reactor (ASBR) with continuous membrane-based liquid-liquid extraction (pertraction). We found that the product spectrum was directed by pH: mildly acidic pH (5-6) led to n-caproate (C6) production, while near-neutral and neutral pH (6.75-7) favored n-caprylate production. In particular, the pH of 6.75 led to the maximum values of volumetric n-caprylate production rate (75.6 {+/-} 0.6 mmol C L-1 d-1; 0.06 g L-1 d-1) and n-caprylate concentration in the fermentation broth (420 mM C; 7.57 g L-1). Given that methane production remained low at near-neutral and neutral pH, we theorized that the high concentration of undissociated n-caprylic acid (5.71 mM C) inhibited methanogenesis. We then demonstrated such an inhibitory effect at neutral pH in: (1) microcosm experiments; and (2) the continuous bioreactor by adding methanogenic sludge. Furthermore, 16S rRNA gene sequencing analysis revealed that near-neutral and neutral pH led to more diverse microbial communities than at mildly-acidic pH. For the first time, we report predominant n-caprylate production in a microbiome at near-neutral and neutral pH conditions where methanogenesis was controlled by the inhibitory effects of undissociated n-caprylic acid. At the same time, extraction of this species occurred even at near-neutral and neutral pH.

bioengineering↗

bioRxiv2020

Food waste as a resource: grinding, dilution, and storage as a pretreatment strategy to produce fermentation intermediates

In several states of the U.S., one measure to mitigate greenhouse-gas emissions has been to ban food wastes from landfills. As a result, U.S.-based companies are now providing decentralized food-waste management systems for supermarkets and restaurants, which include storage as a slurry. It is unclear, however, which storage conditions (factors) would affect the spontaneous microbial activity, resulting in a different fermentation product spectra, and how this would affect further post-treatment. Here, we performed two experiments to mimic: 1) storage and 2) subsequent anaerobic digestion. For the food-waste storage system, we designed a mixed-level fractional factorial analysis with 12 experimental combinations, including separating food waste into: carbohydrate-rich, lipid-rich, and protein-rich food waste. We found that all factors that we tested correlated with the fermentation product spectra, but that especially the factors: i) storage temperature; ii) food-waste composition; and iii) storage time affected the fermentation outcome. We observed that relatively low pH levels of 3-4, which were achieved due to rapid lactic acid accumulation by microbial activity during storage, coincided with greater lactate production at a maximum chemical oxygen demand (COD) selectivity of 90%. This provides an opportunity to optimize lactate production, which is ideal for subsequent methane or chemical production. TOC/Abstract graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗