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Long-term n-caproic acid production from yeast-fermentation beer in an anaerobic bioreactor with continuous product extraction Shijian Ge, Joseph Usack, Catherine M. Spirito, and Largus T. Angenent Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00238 • Publication Date (Web): 05 May 2015 Downloaded from http://pubs.acs.org on May 10, 2015
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Long-term n-caproic acid production from yeast-fermentation beer
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in an anaerobic bioreactor with continuous product extraction
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Shijian Ge, Joseph G. Usack, Catherine M. Spirito, Largus T. Angenent*
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Department of Biological and Environmental Engineering, Cornell University, 226
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Riley-Robb Hall, Ithaca, NY 14853, USA.
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* Corresponding author: E-mail:
[email protected]; Fax: +1-607-255-4449; Tel:
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+1-607-255-2480
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Keywords: n-caproic acid; n-caproate, n-hexanoic acid; carboxylate platform;
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microbiome; chain elongation
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Abstract: Multi-functional reactor microbiomes can elongate short-chain carboxylic
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acids (SCCAs) to medium-chain carboxylic acids (MCCAs), such as n-caproic acid.
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However, it is unclear whether this microbiome biotechnology platform is stable
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enough during long operating periods to consistently produce MCCAs. During a
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period of 550 days, we improved the operating conditions of an anaerobic bioreactor
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for the conversion of complex yeast-fermentation beer from the corn kernel-to-ethanol
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industry into primarily n-caproic acid. We incorporated and improved in-line,
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membrane liquid-liquid extraction to prevent inhibition due to undissociated MCCAs
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at a pH of 5.5 and circumvented the addition of methanogenic inhibitors. The
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microbiome accomplished several functions, including hydrolysis and acidogenesis of
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complex organic compounds and sugars into SCCAs, subsequent chain elongation
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with undistilled ethanol in beer, and hydrogenotrophic methanogenesis. The methane
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yield was 2.40±0.52% based on COD and was limited by the availability of carbon
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dioxide. We achieved an average n-caproate production rate of 3.38±0.42 g l-1 d-1
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(7.52±0.94 g COD l-1 d-1) with an n-caproate yield of 70.3±8.81% and an
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n-caproate/ethanol ratio of 1.19±0.15 based on COD for a period of ~55 days. The
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maximum production rate was achieved by increasing the organic loading rates in
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tandem with elevating the capacity of the extraction system and a change in the
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complex feedstock batch.
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INTRODUCTION
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Studies have estimated that ~20% of the energetic value of ethanol is required for
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distillation in the corn-to-ethanol industry.1 Ethanol distillation is an energy intensive
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process due to the complete miscibility of ethanol and water, which makes ethanol
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separation energetically unfavorable. To overcome this barrier, we have developed a
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fermentation process2 to convert yeast-fermentation beer (unprocessed fermentation
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broth with undistilled, dilute ethanol from the corn kernel-to-ethanol industry) into
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medium-chain carboxylic acids (MCCAs) with 6-12 carbon chains (C6-C12), which
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we refer to here as carboxylates when we discuss product concentrations to identify
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both the undissociated and dissociated species. The undissociated MCCAs are
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extractable from water with much less energy input than ethanol due to their relatively
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low maximum solubilities of 10.8 g L-1 for n-caproic acid (C6) and 0.68 g L-1 for
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n-caprylic acid (C8),3 which makes a selective phase separation process possible.4 In
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addition, MCCAs have higher energy densities than ethanol or n-butyric acid due to
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their higher carbon/oxygen ratio, and they are a superior precursor for further
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processing to biofuels or industrial chemicals via chemical or electrochemical
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downstream processes.2, 5-9
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In our continuous bioprocess, reactor microbiomes (open cultures of microbial
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consortia) sequentially elongate short-chain carboxylic acids (SCCAs), such as acetic
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acid and n-butyric acid, to MCCAs, such as n-caproic acid, by the addition of
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2-carbon (C2) units, which are derived from ethanol that is contained in the
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yeast-fermentation beer. Clostridium kluyveri is one of several bacteria that dominate
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the chain-elongation gene pool in these reactor microbiomes and that utilize the
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reverse β-oxidation pathway for MCCA production.2,
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pathway, which includes ethanol oxidation and reverse β oxidation, is well described
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in the literature with ethanol providing reducing equivalents (NADH2), carbon, and
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energy (ATP) for the C2-chain elongation reaction.11, 14 The yeast cells, C5 sugars,
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and left-over corn grain biomass in the yeast-fermentation beer can also be converted
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to acetic acid or n-butyric acid as the SCCA starting molecule via hydrolysis,
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acidogenesis, and acetogenesis. When the hydrogen partial pressure is high enough in
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the microbiome, the primary fermentation of the yeast-fermentation beer can be
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directed towards n-butyric acid production rather than acetic acid production.15 Then,
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the ethanol in the yeast-fermentation beer can be more efficiently used to elongate
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n-butyric acid to n-caproic acid in a one-step process rather than to elongate in a
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two-step process: 1) acetic acid to n-butyric acid; and then 2) n-butyric acid to
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n-caproic acid. For every five molecules of ethanol that is used for elongation, one
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molecule of ethanol is completely oxidized to acetate for energy. Therefore, additional
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ethanol is necessary for the chain elongation from this produced acetic acid to
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generate n-butyric acid,14 even when only n-butyric acid is being produced from the
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complex organic material in beer.
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The chain elongation
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Steinbusch et al.13 used synthetic acetate and ethanol as substrates to achieve a
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maximum n-caproate production rate of 0.49 g l-1 d-1 (1.09 g COD l-1 d-1) during a
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116-day operating period in which the pH was maintained at 7 without product
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extraction but with addition of the methane inhibitor 2-bromoethanesulfonate. In our
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previously published communication,2 we reported about the first ~120 days of this
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study. We used real yeast-fermentation beer and achieved a short-term maximum
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n-caproate production rate of 2.1 g l-1 d-1 (4.62 g COD l-1 d-1), which was higher than
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the organic loading rate (OLR) of 3.89 g COD l-1 d-1 applied to this system due to
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conversion of accumulated intermediates.2 We included in-line product extraction and
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maintained a pH of 5.5 to: 1) prevent acetogenic methanogenesis without addition of
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2-bromoethanesulfonate; and 2) create a sufficient pH gradient to drive extraction of
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the undissociated n-caproic acid. During the first ~120 days of the operating period,
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the extraction system was limiting the production. The OLR, and thus the production
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rate, could not be increased because accumulating undissociated n-caproic acid at a
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pH of 5.5 would have inhibited the microbial species that perform the
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chain-elongation reaction.16, 17 More recent work by Grootscholten et al.18 suggest that
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the rates of biological chain elongation were not limiting our bioreactor system
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because they achieved a much higher n-caproate production rate of > 50 g l-1 d-1 (110
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g COD l-1 d-1) with synthetic substrate. In a follow-up paper, Grootscholten et al.19
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converted food waste and procured, distilled ethanol into n-caproate in a two-phase
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system. They operated the chain-elongation phase at a neutral pH and short residence
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times of hours to prevent acetoclastic methanogens without the addition of
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2-bromoethanesulfonate18, 19.
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Here, the specific objective was to demonstrate that our chain-elongating microbiome
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biotechnology with a complex organic substrate can be run for long-term operating
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periods with stable performances. To carry out this specific objective, we fed diluted
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yeast-fermentation beer to an anaerobic sequencing batch reactor (ASBR) for an
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operating period exceeding 1.5 years, while gradually increasing the OLR (up to a
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maximum of 10.7 g COD l-1 d-1). Throughout the operating period, we installed an
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improved extraction system to allow for higher organic loading rates and production
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rates. During this change we observed that the microbiome was able to recover from
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disturbances. At the end of the operating period the maximum volumetric n-caproate
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production rate was stable for more than three hydraulic retention time periods (~55
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days).
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MATERIALS AND METHODS
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Bioreactor set-up and extraction system
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A cylindrically shaped, 5-L glass ASBR was operated with a cycle period of 48 h by
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sequencing through four steps: 1) feeding step (3 HRTs); 2) 4.48±0.38 g COD l-1
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d-1, 46.2±3.93%, and 0.78±0.07 during Days 449-483 (>2 HRTs); and 3) 7.52±0.94 g
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COD l-1 d-1, 70.3±8.81%, and 1.19±0.15 during Days 493-547 (>3 HRTs).
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Three operating changes improved the n-caproate production rates
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These improvements in performance during Phase 5 can be explained by the
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following operating changes: i) an increase in the extraction capacity after we had
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increased the pump speed for the oil and stripping solution by ~30% on Day 432; ii) a
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different yeast-fermentation beer batch on Day 445 (Table S-1); and iii) an increase in
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the OLR from 9.71 to 10.7 g COD l-1 d-1 on Day 485 of the operating period (Table 1).
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Before the pump speed change (i) and feedstock change (ii) occurred, the extraction
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system was limiting n-caproate production because an increase in the OLR from 8.87
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to 9.72 COD l-1 d-1 on Day 423 had not increased the performance. After the
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implementation of both changes (i and ii), however, this was reversed and n-caproate
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production rates had become limited by the OLR instead of the extraction rate with an
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average extraction efficiency increase to 98.3% between Days 461-463 (Fig. 2) after
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the effluent n-caproate concentrations had been greatly reduced between Days
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450-475 (Fig. 1B). Because of the OLR limitation, the n-caproate production rates
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had only slightly increased between Days 363-421 and Days 449-483 (student t-test,
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p
