Bioresource Technology 169 (2014) 162–168
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Pre-treatment step with Leuconostoc mesenteroides or L. pseudomesenteroides strains removes furfural from Zymomonas mobilis ethanolic fermentation broth William J. Hunter, Daniel K. Manter ⇑ USDA–ARS, 2150-D Centre Avenue, Fort Collins, CO 80526-8119, USA
h i g h l i g h t s Leuconostoc spp. pre-treatment removes furfural in a model fermentation broth. Leuconostoc spp. consume less than 5% of the available dextrose. Pre-treatment did not reduce the total amount of ethanol production. Pre-treatment and fermentation could be conducted in a single vessel.
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Article history: Received 6 May 2014 Received in revised form 25 June 2014 Accepted 26 June 2014 Available online 3 July 2014 Keywords: Furan-2-carboxaldehyde 2-Furaldehyde Furfuraldehyde Biofuel Furfural alcohol
a b s t r a c t Furfural is an inhibitor of growth and ethanol production by Zymomonas mobilis. This study used a naturally occurring (not GMO) biological pre-treatment to reduce that amount of furfural in a model fermentation broth. Pre-treatment involved inoculating and incubating the fermentation broth with strains of Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides. The Leuconostoc strains converted furfural to furfuryl alcohol without consuming large amounts of dextrose in the process. Coupling this pre-treatment to ethanolic fermentation reduced furfural in the broth and improved growth, dextrose uptake and ethanol formation. Pre-treatment permitted ethanol formation in the presence of 5.2 g L1 furfural, which was otherwise inhibitive. The pre-treatment and presence of the Leuconostoc strains in the fermentation broth did not interfere with Z. mobilis ethanolic fermentation or the amounts of ethanol produced. The method suggests a possible technique for reducing the effect that furfural has on the production of ethanol for use as a biofuel. Published by Elsevier Ltd.
1. Introduction Fossil fuel resources are limited and their supply can be disrupted during international conflicts. In addition, air, soil, and water are degraded by the burning of these fuels or during their extraction, transport and storage; greenhouse gases are a particular point of concern associated with the use of fossil fuels (Solomon, 2007; Zecca and Chiari, 2010). It is estimated that damage caused by fossil fuel use equals 14% of Gross World Product (Barbir et al., 1990). Due to these concerns alternative sources of liquid fuels are needed (Silveira and Khatiwada, 2010; Lubbe and Sahlin, 2012). One promising alternative involves the conversion of biomass cellulose to ethanol for fuel. Biomass is usually a low cost renewable material made up of agricultural residues, ⇑ Corresponding author. Tel.: +1 970 492 7255. E-mail address: [email protected] (D.K. Manter). http://dx.doi.org/10.1016/j.biortech.2014.06.097 0960-8524/Published by Elsevier Ltd.
dedicated fuel crops, or wood. However, the structural aspect of biomass that makes it resistant to microbial attack also hinders the use of these materials as a carbon source for microbial ethanolic fermentation. To enhance the fermentation process, biomass is typically exposed to a pretreatment step(s) in order to alter the structural characteristics of lignocellulose and make cellulose more accessible for hydrolysis (Alvira et al., 2010). The most common and most economical method of hydrolysis uses heat and dilute (0.5–3.0 wt.%) sulfuric acid (Torget et al., 1991). This critical step opens up the biomass structure exposing the cellulose, thus permitting enzymatic digestion (Lloyd and Wyman, 2005). There are, however, problems associated with the hydrolysis step. One involves the formation of compounds (i) weak acids, (ii) phenolic compounds, and (iii) furan aldehydes that interfere with the ethanolic fermentation (Okuda et al., 2008). Of these it is the furan aldehydes, 5-(hydroxyl methyl)-2-furaldehyde and furfural that are the most significant inhibitors of growth and ethanol
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production by microorganisms that ferment sugars to ethanol (Delgenes et al., 1996; Chandel et al., 2013). Chemical as well as biological approaches can remove the furan aldehydes. Chemical approaches include the most used method, overliming or treatment of the hydrolysate with calcium hydroxide. It is a cost-effective method but it also results in the loss of about 10% of the sugar (Palmqvist et al., 1996; Martinez et al., 2000, 2001). Ion exchange resins, electrodialysis, nanofiltration and activated carbon treatment can also remove the furan aldehydes from hydrolysates but these processes can be expensive and can sometimes result in a loss of sugar (Mussatto and Roberto, 2004; Sainio et al., 2011; Lee et al., 2014; Brás et al., 2014). Biological approaches have also been used that employed a variety of microorganisms to remove furfural or other inhibitors. Organisms that have been studied include Acinetobacter sp., Arthrobacter aurescens, Flavobacterium indologenes, Methylobacterium extorquens, Pseudomonas sp., Ureibacillus thermosphaericus, Trametes versicolor, Trichoderma reesei, and several unidentified yeast (Chandel et al., 2013); however, many of these organisms consume significant amounts (25–73%) of sugar in the process (e.g., Ran et al., 2014). This study concentrates on the furan aldehyde furfural and the impact that it has on growth and ethanolic fermentation by the bacteria Zymomonas mobilis. The objective of the present study was to examine the use of Leuconostoc mesenteroides strains RR and WCF2 and Leuconostoc pseudomesenteroides strain WCF3 as in situ microbial detoxification agents to remove furfural from a simulated Z. mobilis ethanolic fermentation broth. For this study the detoxification process and the ethanolic fermentation process took place sequentially in the same vessel, a desirable approach (Delgenes et al., 1996; Chandel et al., 2013) and involved a two stage process. The first stage consisted of a biological detoxification stage (Stage 1) that used L. mesenteroides or L. pseudomesenteroides strains to degrade furfural, Stage 1 was followed by a fermentation stage (Stage 2) that used Z. mobilis to ferment dextrose to ethanol. 2. Methods 2.1. Media and incubation conditions The AmPSY base medium, pH 5.3, contained 1.2 g (NH4)2SO4, 10 g yeast extract, and 0.6 g (NH4)2PO4 L1. HM/RSM base medium, pH 6.8, contained 1.3 g N-2-hydroxyethylpiperazine-N0 -ethanesulfonic acid, 1.1 g 2-(N-morpholino)ethanesulfonic acid, 6.5 g yeast extract, 1 g (NH4)2SO4, 0.02 g KH2PO4, 0.5 g MgSO4 L1. Dextrose or furfural was added to the base media when indicated. Glassware was sterilized by autoclaving for 15 min at 121 °C. Broth media were filter sterilized (0.22 lm, Millipore, Bedford, MA) and added to the autoclaved glassware. Agar media was prepared by supplementing broth media with 1.5% (w/v) Bacto-Agar. Agar media was sterilized by autoclaving at 121 °C for 15 min. Studies were carried out in 50 ml long neck culture flasks containing 30 ml of HM-RSM base media supplemented with furfural as indicated and with 15% dextrose unless otherwise indicated. Flasks were closed with Kimble PM Caps (Rockwood, TN, USA). All incubations were at 30 °C, aerobic incubations were shaken at 100 rpm while fermentative incubations were stationary. Unless otherwise indicated inoculum was 6 ml of late log phase culture grown in the same media used in the study but without furfural. 2.2. Microorganisms Z. mobilis strain ATCCÒ 31821™ (Rhee et al., 1984) was obtained from the American Type Culture Collection. New bacterial strains that could tolerate high levels of furfural were isolated from
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various sources (compost or decaying railroad ties) as follows. Source material was diluted 1:1 (weight:volume) into sterile saline and streaked onto AmPSY agar plates supplemented with 6 g L1 furfural and 0.1 g L1 cycloheximide and incubated until colonies developed (4 days). Individual colonies were selected and tested for their ability to degrade furfural (data not shown). Based on this initial screening, three isolates (WCF2 and WCF3 isolated from compost, RR isolated from a decaying railroad tie) were selected for use in this study. All cultures were stored at 4 °C in AmPSY broth media supplemented with 3 g L1 dextrose. 2.3. Identification of strain RR, strain WCF2, and WCF3 The three newly isolated bacterial strains were identified as L. mesenteroides strains RR and WCF2 and L. pseudomesenteroides strain WCF3 based on sequencing of the 16S rRNA gene. Briefly, genomic DNA was isolated from pure cultures and 10 ng was amplified with the 27F and 1492R (Lane, 1991) primers. The resultant amplicon was cloned using the TOPO TA cloning kit for sequencing (Invitrogen, Carlsbad, CA) and sequenced in both directions using Big Dye cycle sequencing chemistry (Applied Biosystems, Carlsbad, CA). For the construction of a UPGMA tree (Fig. 1), sequences of related type strains were extracted from GenBank, aligned with ClustalW, and a UPGMA tree constructed using the MEGA 5.0 computer program (http://www.megasoft.net/) (Tamura et al., 2011). The 16S rRNA gene sequence for the WCF3 strain was deposited as accession KF698922 into the GenBank database and a culture of L. pseudomesenteroides strain WCF3 was deposited in the US Department of Agriculture, Agricultural Research Service (NRRL) Culture Collection as NRRL B-59999. 2.4. Studies The first study, a preliminary study, investigated the influence furfural had on Z. mobilis cell growth and ethanol production. The study consisted of two treatment groups and a control group, all incubated under fermentative conditions. The control group received no furfural and the two treatments groups received 1.9 and 4.3 g L1 furfural. Samples of the culture fluid were collected at the intervals indicated and assayed (see below) for growth and ethanol production. A second study, consisting of four treatment groups incubated aerobically, investigated the influence 0, 2.2, 3.5 and 4.5 g L1 amounts of furfural had on growth and furfural degradation by the three Leuconostoc strains RR, WCF2 and WCF3. Samples of the culture fluid were collected at the intervals indicated and assayed for growth and furfural content. Study three, an aerobic study, investigated the rate of furfural degradation by the three Leuconostoc strains and the principal furfural degradation product or products produced during the incubation. Only one level of furfural, 5.2 g L1 was evaluated; other conditions were as described above for study #2. Samples were collected and assayed by liquid chromatography (see below) for furfural and possible furfural degradation products. For this study, the 6 ml late log inoculum was concentrated 10-fold via centrifugation at 1620g for 15 min. The fourth and final study investigated the influence of Leuconostoc strains RR, WCF2 and WCF3 pre-treatments on the growth and ethanolic fermentation of Z. mobilis. Stage 1 incubations, the pre-treatment, were aerobic for 4 days. Stage 1 flasks were inoculated with 6 mls of late log phase cultures of the RR, WCF2 or WCF3 strains grown in HM/RSM base media supplemented with 3% dextrose; controls received 6 ml of uninoculated HM/RSM base media supplemented dextrose. Experimental media for Stage 1 contained 15% dextrose and furfural concentrations from 1.9 to 5.2 mM. For Stage 2 incubations, the ethanolic fermentation stage, the flasks
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WCF2 53 gi|116096021|L. mesenteroides subsp. mesenteroides ATCC8293T 51
gi|631252056|L. mesenteroides subsp. dextranicum JCM9700T gb|CP000414.1|:22669-24217 L. mesenteroides subsp. mesenteroides ATCC8293T
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gi|11602803|L. mesenteroides subsp. cremoris NCFB543T gi|175133|L.mesenteroides DSM20343T
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WCF3 58 50
gi|317016875|L. pseudomesenteroides KCTC3652T gi|6683524|L. pseudomesenteroides NRIC1777T gi|221675373|L. gasicomitatum LMG18811T gi|209413803|L. palmae TMW2.694T
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Fig. 1. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 0.04082069 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. Evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1402 positions in the final dataset.
from Stage 1, after 4 days of pre-treatment, were inoculated with cells from a 3 day old culture of Z. mobilis. The Z. mobilis inoculum cells in 6 ml of RM/RSM broth supplemented with dextrose were pelleted via centrifugation at 1620g for 15 min and the pelleted cells resuspended in 6 mls of culture fluid from one of the Stage 1 pre-treatment cultures. The culture fluid plus Z. mobilis cells were added back to the original culture. Stage 2 incubations were for an additional 11 days with no shaking. Samples were collected at intervals and assayed for furfural, cell growth, dextrose content and ethanol content. 2.5. Chemicals Furfural (99% pure) was purchased from Sigma–Aldrich (St. Louis, MO); ethyl alcohol (200 proof) was obtained from Aaper Alcohol and Chemical Co. (Shelbyville, KY), and dextrose (ACS grade) was purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals were reagent grade. 2.6. Analysis Samples of the cultures (1 ml) were collected at the indicated intervals, diluted with DI water (1:9) and optical density (OD) measured at a wavelength of 660 nm. Next the diluted samples were centrifuged through Ultrafree™ 0.22 lm centrifuge filters (Millipore, Bedford, MA) spun at 10,000g for 5 min and the filtrates analyzed for dextrose, ethanol and furfural content. Furfural was estimated via a high pressure liquid chromatography (HPLC) equipped with a C-18 column and a photodiode array detector as described earlier (Hunter et al., 2012). Samples for ethanol and dextrose analysis were diluted with additional water 1:9 (v:v). Dextrose and ethanol were measured via a HPLC (LC10, Shimadzu Scientific Instruments, Columbia, MD) equipped with an AminexÒ HPX-87P 300 by 7.8 mm column (Bio-Rad Labs, Richmond, CA), a refractive index detector (RID-10A, Shimadzu) and an evaporative light scattering detector (ELSD-LTII, Shimadzu) operated at 60 °C and 360 kPa nitrogen. Injection volume was 20 ll, column temperature was 85 °C, and the mobile phase was water (Millipore MilliQ, Millipore Corp., Bedford, MA) pumped at 0.60 ml min1. Alternatively, ethanol was also measured via a gas chromatograph (GC17, Shimadzu) equipped with an Alltech Econocap ECwax column (Grace, Deerfield, IL) operated at 105 °C and with a flame ionization detector.
2.7. Statistics All controls and treatment groups consisted of three independent replicates. All data are presented as mean and standard error of the mean (SEM). For each timepoint (day) of interest, statistical significance between treatments was computed using a one-way ANOVA with the InstatÒ computer program (GraphPad Software Inc., San Diego, CA), post hoc tests (Bonferroni-corrected) were considered statistically significant for p-values below 0.05. Ethanol production rates (g L1 h1) were calculated between consecutive timepoints (e.g., Day4–Day2, Day7–Day4) using the mean treatment values (n = 3), and the highest value for each treatment, regardless of time period, is reported as the maximum ethanol production rate. 3. Results and discussion 3.1. Influence of furfural on Z. mobilis – a preliminary study Furfural, as has been shown previously (Chandel et al., 2013), had a detrimental influence on Z. mobilis growth. In this study, growth on day 1 was significantly reduced by 35% and 84% for the 1.9 g L1 and 4.3 g L1 furfural treatments, respectively (Fig. 2A). Interestingly, total ethanol production was not reduced by the two treatments though the rate at which it was produced was slowed. For example, the amount of ethanol on day 1 was significantly reduced by 33% and 92% for the 1.9 g L1 and 4.3 g L1 furfural treatments, respectively; however, by day 2 (1.9 g L1 furfural) and 7 (4.3 g L1 furfural), the total amount of ethanol present was equal to the control levels. The results show that furfural, at these levels, interfered with the Z. mobilis growth and the rate of ethanol production, but not the total amount of ethanol that could be produced. 3.2. Furfural degradation by the L. mesenteroides and pseudomesenteroides strains A second study examined the ability of the RR, WCF2 and WCF3 strains to grow in the presence of furfural as well as the ability of these L. mesenteroides and pseudomesenteroides strains to degrade furfural. The furfural treatments slowed the growth rates of all three strains, but the amounts of growth inhibition were not large (Fig. 3A–C). On day 2, the RR strain’s growth was inhibited 11–20%
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Growth (A660)
These studies showed that all three L. mesenteroides and L. pseudomesenteroides strains grew well in the presence of furfural and that all were capable of removing large amounts of furfural from the fermentation broths.
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3.3. Conversion of furfural to furfural-OH by the L. mesenteroides and L. pseudomesenteroides strains
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A third study looked at the break down products that accumulate during the degradation of furfural by the L. mesenteroides and L. pseudomesenteroides strains. Many different bacteria transform or degrade furfural and a number of different pathways are used by these bacteria. One of the simplest transformations involves the reduction of the aldehyde group to an alcohol group (Wierckx et al., 2011) yielding furfuryl alcohol (furfuryl-OH). This is a desirable transformation as the aldehyde group is more toxic than is the alcohol group. HPLC analysis showed that 89 ± 1.2% of the furfural that was lost from the incubations accumulated as furfural-OH and that furfural disappeared from the incubations at a maximum rate of 2.7 g L1 day1 (Fig. 4). Of the three strains evaluated, the WCF3 strain performed best in this study though the differences between strains were small. The formation of furfural-OH in the fermentation broth, though yields are low, might present a market opportunity. Both furfural and furfural-OH have value as industrial chemicals. Furfural-OH is used as a flavoring agent and as a fragrance; it is also used in the formation of thermosetting resins, in the production of tetrahydrofurfuryl alcohol, a chemical used as an adjuvant in herbicide formulations, and as a nonhazardous solvent (Adams et al., 1997; Oliveira et al., 2008; Song et al., 2007). Furfuryl-OH has a 2014 market price of about $1200–3000 metric ton1 in bulk while furfural has a market price of $1000–1900 metric ton1 in bulk according to Alibaba.com, an online marketplace for international trade. 3.4. Use of the L. mesenteroides and pseudomesenteroides strains as a biological pre-treatment to remove furfural A forth study evaluated the use of the L. mesenteroides and L. pseudomesenteroides strains as a method for removing furfural from the fermentation broth before the start of a Z. mobilis ethanolic fermentation. For this study furfural treatment levels of 1.9, 3.0, 4.2, and 5.2 g L1 were used with each of the three L.
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by the three furfural treatments, but by day 4 these cultures had recovered and there were no significant differences between treatments (Fig. 3A). A somewhat similar pattern (Fig. 3B) was seen with the WCF2 strain where treatment inhibitions of 18–23% were observed on day 2, but by day 4 the treatment inhibitions had declined to 0–10%. WCF3 growth, which was less than that observed with the other two strains, followed the same pattern (Fig. 3C), with day 2 growth being inhibited 15–24% by the treatments and day 4 growth inhibited by 12–18%. With all three strains, furfural levels in the culture fluid decreased during the incubations. The largest and most rapid decreases were observed with the RR strain where, by day 4, furfural levels ranged from 0.0 to 0.91 g L1 (Fig. 3D). With the WCF2 and WCF3 strains furfural levels decreased to 0.0 to 1.4 and 0.0 to 1.6 g L1, respectively, by day 4 (Fig. 3E and F).
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Fig. 3. The ability of L. mesenteroides and pseudomesenteroides strains to grow in the presence of furfural (A, B, and C) and their ability to degrade furfural (D, E, and F).
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RR Maximum degradation rate = 2.7 ± 0.07 g L-1 Day-1
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Fig. 4. The conversion of furfural to furfural-OH by L. mesenteroides and pseudomesenteroides strains.
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mesenteroides and L. pseudomesenteroides strains. During the Stage 1 pre-treatment the three strains RR, WCF2 and WCF3 removed >99% of the 1.9 g L1 furfural initially present, 86–99% of the 3.0 g L1 furfural initially present, 81–90% of the 4.2 g L1 furfural initially present, and 62–66% of the 5.2 g L1 furfural initially present (Fig. 5A–D); thereby, significantly reducing the amount of furfural present at the start of the ethanolic fermentation stage. The uninoculated negative control confirmed that the furfural was not lost due to volatilization, degradation, etc. The negative control data also showed that Z. mobilis alone was able to degrade furfural during Stage 2 (ethanolic fermentation stage); and by day 2, Z. mobilis had degraded 95% of the furfural from the 1.9, 3.0 and 4.2 g L1 furfural treatments (Fig. 5A–C), but only 86% of the furfural from the 5.2 g L1 furfural treatment level (Fig. 5D). At the end of the study (day 11), the Z. mobilis cells had removed only 85% of the furfural (i.e., 0.73 g L1 remaining); whereas, all three Leuconostoc strains had removed 100%.
increased Z. mobilis growth on day 2 by 85% and on day 4 by 65%, almost reversing the inhibition caused by furfural on these days (Fig. 5E). In contrast, the WCF2 and RR strains only increased Z. mobilis growth on these days by averages of 45% and 6%, respectively. At the higher amounts of furfural the differences between the L. mesenteroides and L. pseudomesenteroides strains became less noticeable, and in no instance did the pre-treatment restore full Z. mobilis growth (e.g., similar to that of the positive controls) . 3.6. Impact of furfural and the pretreatments on Z. mobilis dextrose uptake Dextrose utilization by all three Leuconostoc strains was very low during the Stage 1 pre-treatments, but appeared to increase with increasing furfural concentrations. For example, the average dextrose consumption by the three Leuconostoc strains was 3.8%, 2.1%, 5.1%, and 16.4% for the 1.9, 2.0, 4.2, and 5.9 g L1 treatments, respectively (Fig. 6A–D). The Stage 1 pre-treatments had a clear impact on dextrose uptake during the Stage 2 ethanolic fermentations. Furfural had a progressive and negative impact on dextrose uptake by Z. mobilis when no L. mesenteroides and L. pseudomesenteroides pre-treatments were employed (Fig. 6A–D). For the 1.9 g L1 furfural treatment, no significant differences in Z. mobilis dextrose consumption were observed, with dextrose consumption nearly complete (> 90%) by day 4 for all of the treatments (Fig. 6A). However, for the 3.0 and 4.2 g L1 treatments (Fig. 6B and C) dextrose consumption was nearly complete (>90% consumed) by day 4 for all three Leuconostoc pre-treatments, but not until day 7 for the negative control. The 5.2 g L1 treatment essentially prevented dextrose uptake in the negative control (Fig. 6D), which could be restored by pre-treatment with all three Leuconostoc strains; however, dextrose consumption was still delayed (i.e., >90% consumption at day 7).
3.5. Impact of furfural and the L. mesenteroides and pseudomesenteroides pre-treatment on Z. mobilis cell growth As was seen in the preliminary study (Fig. 2A) a comparison of data from the positive control (no furfural) and negative control (furfural but no L. mesenteroides or L. pseudomesenteroides pretreatment) shows that the presence of furfural in the fermentation media reduced the growth of Z. mobilis cells (Fig. 5E–H). The growth inhibition of Z. mobilis caused by the furfural treatments was reduced by the Stage 1 pre-treatments, with all three of the L. mesenteroides and L. pseudomesenteroides strains improving the growth of Z. mobilis despite the high level of furfural in the original media. The WCF3 strain preformed best in most of these studies followed by the WCF2 strain and then by the RR strain. At the 1.9 g L1 furfural level the WCF3 Stage 1 pre-treatment
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Fig. 5. Furfural concentration and cell growth during the two stage incubations. The first portion (Stage 1) of the incubation was an aerobic 4 day pre-treatment with no inoculum, the control, or with one of three L. mesenteroides or pseudomesenteroides strains as the inoculum. The second portion (Stage 2) of the incubation, lasting from day 0 to day 11, was an ethanolic fermentation. All incubation flasks were inoculated with Z. mobilis on day 0.
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Fig. 6. Dextrose and ethanol concentration during the two stage incubations. The Stage 1 incubation was an aerobic 4 day pre-fermentation incubation with no inoculum or with one of three L. mesenteroides or pseudomesenteroides strains as the inoculum. The Stage 2 incubation, lasting from day 0 to day 11, was a fermentative incubation. All incubation flasks were inoculated with Z. mobilis on day 0.
Most importantly, during the pre-treatment all three Leuconostoc strains did not appear to consume large amounts of dextrose. The average loss of dextrose, across all treatments, was only about 5% during the 4 day pre-treatment incubation. 3.7. Impact of furfural and the pretreatments on ethanol production As was the case in the preliminary study (Fig. 2B), furfural had a clear negative impact on ethanol production by Z. mobilis; however, the Stage 1 pre-treatments with the L. mesenteroides and L. pseudomesenteroides strains were able to partially alleviate the inhibition caused by furfural. Without the pre-treatment, positive controls vs negative controls (Fig. 6E–H), furfural had a generally progressive and negative impact on ethanol production during the Stage 2 fermentations. At the 1.9 g L1 level furfural delayed the onset of ethanol production by about a day and reduced the maximum rate of ethanol production by about 42% (Fig. 6E), from 1.34 to 0.76 g L1 h1; while the 3.0 and 4.2 g L1 treatments (Fig. 6F and G) delayed the onset of ethanol production by 2 and 4 days, respectively, and retarded production by 70%. The highest furfural treatment level, 5.2 g L1 essentially stopped ethanolic fermentation (Fig. 6H). The L. mesenteroides and L. pseudomesenteroides pretreatments reduced the inhibition caused by furfural and increased the rate of ethanol production relative to the negative controls. Pretreatments with the RR, WCF2 and WCF3 strains increased the maximum ethanol production rates in the 1.9 g L1 furfural treatment group to 0.98, 1.60, and 2.19 g L1 h1, respectively. In the 3.0 g L1 furfural treatment group the negative control rate was 0.55 g L1 h1 and the fermentations that were pretreated with RR, WCF2 and WCF3 in Stage 1 had maximum ethanol production rates of 1.18, 1.43, and 1.86 g L1 h1, respectively. In the 4.2 g L1 furfural treatment group the negative control rate was 0.67 g L1 h1 and the fermentations that were pretreated with RR, WCF2
and WCF3 in Stage 1 had production rates of 0.92, 0.77, and 1.26 g L1 h1, respectively. In the 5.2 g L1 furfural treatment group the negative control rate was 0.05 g L1 h1 and the fermentations that were pretreated with RR, WCF2 and WCF3 in Stage 1 formed ethanol at maximum rates of 0.72, 0.66 and 0.56 g L1 h1, respectively. 4. Conclusions Furfural greatly reduced or prevented growth and ethanol production by Z. mobilis cells but not the growth of Leuconostoc cells. Moreover, Leuconostoc cells degraded furfural to the less toxic furfural-OH. A biological pre-treatment of a model fermentation broth with Leuconostoc cells reduced the furfural in the broth improving Z. mobilis growth and, most importantly, ethanol production. The Leuconostoc strains did not consume large amounts of dextrose during the pre-treatment and did not significantly reduce the total amount of ethanol formed, relative to non-furfural containing controls. Additionally, the furfural-OH formed by the Leuconostoc cells has commercial value. Acknowledgements The authors thank Robin Montenieri and Joshua Padilla for their skilled technical support. Manufacturer and product brand names are given for the reader’s convenience and do not reflect endorsement by the US government. This article was the work of US government employees engaged in their official duties and is exempt from copyright. References Adams, T.B., Doull, J., Goodman, J.I., Munro, I.C., Newberne, P., Portoghese, P.S., Smith, R.L., Wagner, B.M., Weil, C.S., Woods, L.A., Ford, R.A., 1997. The FEMA
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GRAS assessment of furfural used as a flavor ingredient. Flavor and Extract Manufacturers’ Association. Food Chem. Toxicol. 35 (8), 739–751. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861. Barbir, F., Veziroglu, T.N., Plass Jr., H.J., 1990. Environmental damage due to fossil fuels use. Int. J. Hydrogen Energy 15, 739–749. Brás, T., Guerra, V., Torrado, I., Lourenço, P., Carvalheiro, F., Duarte, L.C., Neves, L.A., 2014. Detoxification of hemicellulosic hydrolysates from extracted olive pomace by diananofiltration. Process Biochem. 49, 173–180. Chandel, A.K., da Silva, S.S., Singh, O.V., 2013. Detoxification of lignocellulose hydrolysates: biochemical and metabolic engineering toward white biotechnology. Bioenergy Res. 6, 388–401. Delgenes, J.P., Moletta, R., Navarro, J.M., 1996. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb. Technol. 19, 220–225. Hunter, W.J., Manter, D.K., van der Lelie, D., 2012. Biotransformation of ferulic acid to 4-vinylguaiacol by Enterobacter soli and E. aerogenes. Curr. Microbiol. 65, 752–757. Lane, D.J., 1991. RNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York, pp. 115–147. Lee, J.-W., Trinh, L.T.P., Lee, H.-J., 2014. Removal of inhibitors from a hydrolysate of lignocellulosic biomass using electrodialysis. Sep. Purif. Technol. 122, 242–247. Lloyd, T.A., Wyman, C.E., 2005. Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids. Bioresour. Technol. 96, 1967–1977. Lubbe, N., Sahlin, U., 2012. Benefits of biofuels in Sweden: a probabilistic reassessment of the index of new cars’ climate impact. Appl. Energy 92, 473–479. Martinez, A., Rodriguez, M.E., York, S.W., Preston, J.F., Ingram, L.O., 2000. Effect of Ca(OH) treatments (‘‘overliming’’) on the composition and toxicity of bagasse hemicellulose hydrolysates. Biotechnol. Bioeng. 69, 526–536. Martinez, A., Rodriguez, M.E., Wells, M.L., York, S.W., Preston, J.F., Ingram, L.O., 2001. Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol. Prog. 17, 287–293. Mussatto, S.I., Roberto, I.C., 2004. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresour. Technol. 93, 1–10.
Okuda, N., Soneura, M., Ninomiya, K., Katakura, Y., Shioya, S., 2008. Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production. J. Biosci. Bioeng. 106, 128–133. Oliveira, F.B., Gardrat, C., Enjalbal, C., Frollini, E., Castellan, A., 2008. Phenol-furfural resins to elaborate composites reinforced with sisal fibers-molecular analysis of resin and properties of composites. J. Appl. Polym. Sci. 109, 2291–2303. Palmqvist, E., Hahn-Hiigerdal, B., Galbe, M., Zacchi, G., 1996. The effect of watersoluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation. Enzyme Microb. Technol. 19, 470–476. Ran, H., Zhang, J., Gao, Q., Lin, Z., Bao, J., 2014. Analysis of biodegradation performance of furfural and 5-hydroxymethylfurfural by Amorphotheca resinae ZN1. Biotechnol. Biofuels 7, 51. Rhee, S.K., Pagan, R.J., Lefebvre, M.F., Wong, L., Rogers, P.L., 1984. Ethanol production from desalted molasses using Saccharomyces uvarum and Zymomonas mobilis. J. Ferment. Technol. 62, 297–300. Sainio, T., Turku, I., Heinonen, J., 2011. Adsorptive removal of fermentation inhibitors from concentrated acid hydrolyzates of lignocellulosic biomass. Bioresour. Technol. 102, 6048–6057. Silveira, S., Khatiwada, D., 2010. Ethanol production and fuel substitution in Nepal— opportunity to promote sustainable development and climate change mitigation. Renew. Sustain. Energy Rev. 14, 1644–1652. Solomon, S., 2007. Intergovernmental Panel on Climate Change. Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, New York. Song, Y., Li, W., Zhang, M., Tao, K., 2007. Hydrogenation of furfuryl alcohol to tetrahydrofurfuryl alcohol on NiB/SiO amorphous alloy catalyst. Front. Chem. Eng. China 1 (2), 151–154. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony method. Mol. Biol. Evol. 28, 2731–2739. Torget, R., Walter, P., Himmel, M., Grohmann, K., 1991. Dilute-acid pretreatment of corn residues and short-rotation woody crops. Appl. Biochem. Biotechnol. 28– 29, 75–86. Wierckx, N., Koopman, F., Ruijssenaars, H.J., de Winde, J.H., 2011. Microbial degradation of furanic compounds: biochemistry, genetics, and impact. Appl. Microbiol. Biotechnol. 92, 1095–1105. Zecca, A., Chiari, L., 2010. Fossil-fuel constraints on global warming. Energy Policy 38, 1–3.
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Pre-treatment step with Leuconostoc mesenteroides or L. pseudomesenteroides strains removes furfural from Zymomonas mobilis ethanolic fermentation broth William J. Hunter, Daniel K. Manter ⇑ USDA–ARS, 2150-D Centre Avenue, Fort Collins, CO 80526-8119, USA
h i g h l i g h t s Leuconostoc spp. pre-treatment removes furfural in a model fermentation broth. Leuconostoc spp. consume less than 5% of the available dextrose. Pre-treatment did not reduce the total amount of ethanol production. Pre-treatment and fermentation could be conducted in a single vessel.
a r t i c l e
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Article history: Received 6 May 2014 Received in revised form 25 June 2014 Accepted 26 June 2014 Available online 3 July 2014 Keywords: Furan-2-carboxaldehyde 2-Furaldehyde Furfuraldehyde Biofuel Furfural alcohol
a b s t r a c t Furfural is an inhibitor of growth and ethanol production by Zymomonas mobilis. This study used a naturally occurring (not GMO) biological pre-treatment to reduce that amount of furfural in a model fermentation broth. Pre-treatment involved inoculating and incubating the fermentation broth with strains of Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides. The Leuconostoc strains converted furfural to furfuryl alcohol without consuming large amounts of dextrose in the process. Coupling this pre-treatment to ethanolic fermentation reduced furfural in the broth and improved growth, dextrose uptake and ethanol formation. Pre-treatment permitted ethanol formation in the presence of 5.2 g L1 furfural, which was otherwise inhibitive. The pre-treatment and presence of the Leuconostoc strains in the fermentation broth did not interfere with Z. mobilis ethanolic fermentation or the amounts of ethanol produced. The method suggests a possible technique for reducing the effect that furfural has on the production of ethanol for use as a biofuel. Published by Elsevier Ltd.
1. Introduction Fossil fuel resources are limited and their supply can be disrupted during international conflicts. In addition, air, soil, and water are degraded by the burning of these fuels or during their extraction, transport and storage; greenhouse gases are a particular point of concern associated with the use of fossil fuels (Solomon, 2007; Zecca and Chiari, 2010). It is estimated that damage caused by fossil fuel use equals 14% of Gross World Product (Barbir et al., 1990). Due to these concerns alternative sources of liquid fuels are needed (Silveira and Khatiwada, 2010; Lubbe and Sahlin, 2012). One promising alternative involves the conversion of biomass cellulose to ethanol for fuel. Biomass is usually a low cost renewable material made up of agricultural residues, ⇑ Corresponding author. Tel.: +1 970 492 7255. E-mail address: [email protected] (D.K. Manter). http://dx.doi.org/10.1016/j.biortech.2014.06.097 0960-8524/Published by Elsevier Ltd.
dedicated fuel crops, or wood. However, the structural aspect of biomass that makes it resistant to microbial attack also hinders the use of these materials as a carbon source for microbial ethanolic fermentation. To enhance the fermentation process, biomass is typically exposed to a pretreatment step(s) in order to alter the structural characteristics of lignocellulose and make cellulose more accessible for hydrolysis (Alvira et al., 2010). The most common and most economical method of hydrolysis uses heat and dilute (0.5–3.0 wt.%) sulfuric acid (Torget et al., 1991). This critical step opens up the biomass structure exposing the cellulose, thus permitting enzymatic digestion (Lloyd and Wyman, 2005). There are, however, problems associated with the hydrolysis step. One involves the formation of compounds (i) weak acids, (ii) phenolic compounds, and (iii) furan aldehydes that interfere with the ethanolic fermentation (Okuda et al., 2008). Of these it is the furan aldehydes, 5-(hydroxyl methyl)-2-furaldehyde and furfural that are the most significant inhibitors of growth and ethanol
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production by microorganisms that ferment sugars to ethanol (Delgenes et al., 1996; Chandel et al., 2013). Chemical as well as biological approaches can remove the furan aldehydes. Chemical approaches include the most used method, overliming or treatment of the hydrolysate with calcium hydroxide. It is a cost-effective method but it also results in the loss of about 10% of the sugar (Palmqvist et al., 1996; Martinez et al., 2000, 2001). Ion exchange resins, electrodialysis, nanofiltration and activated carbon treatment can also remove the furan aldehydes from hydrolysates but these processes can be expensive and can sometimes result in a loss of sugar (Mussatto and Roberto, 2004; Sainio et al., 2011; Lee et al., 2014; Brás et al., 2014). Biological approaches have also been used that employed a variety of microorganisms to remove furfural or other inhibitors. Organisms that have been studied include Acinetobacter sp., Arthrobacter aurescens, Flavobacterium indologenes, Methylobacterium extorquens, Pseudomonas sp., Ureibacillus thermosphaericus, Trametes versicolor, Trichoderma reesei, and several unidentified yeast (Chandel et al., 2013); however, many of these organisms consume significant amounts (25–73%) of sugar in the process (e.g., Ran et al., 2014). This study concentrates on the furan aldehyde furfural and the impact that it has on growth and ethanolic fermentation by the bacteria Zymomonas mobilis. The objective of the present study was to examine the use of Leuconostoc mesenteroides strains RR and WCF2 and Leuconostoc pseudomesenteroides strain WCF3 as in situ microbial detoxification agents to remove furfural from a simulated Z. mobilis ethanolic fermentation broth. For this study the detoxification process and the ethanolic fermentation process took place sequentially in the same vessel, a desirable approach (Delgenes et al., 1996; Chandel et al., 2013) and involved a two stage process. The first stage consisted of a biological detoxification stage (Stage 1) that used L. mesenteroides or L. pseudomesenteroides strains to degrade furfural, Stage 1 was followed by a fermentation stage (Stage 2) that used Z. mobilis to ferment dextrose to ethanol. 2. Methods 2.1. Media and incubation conditions The AmPSY base medium, pH 5.3, contained 1.2 g (NH4)2SO4, 10 g yeast extract, and 0.6 g (NH4)2PO4 L1. HM/RSM base medium, pH 6.8, contained 1.3 g N-2-hydroxyethylpiperazine-N0 -ethanesulfonic acid, 1.1 g 2-(N-morpholino)ethanesulfonic acid, 6.5 g yeast extract, 1 g (NH4)2SO4, 0.02 g KH2PO4, 0.5 g MgSO4 L1. Dextrose or furfural was added to the base media when indicated. Glassware was sterilized by autoclaving for 15 min at 121 °C. Broth media were filter sterilized (0.22 lm, Millipore, Bedford, MA) and added to the autoclaved glassware. Agar media was prepared by supplementing broth media with 1.5% (w/v) Bacto-Agar. Agar media was sterilized by autoclaving at 121 °C for 15 min. Studies were carried out in 50 ml long neck culture flasks containing 30 ml of HM-RSM base media supplemented with furfural as indicated and with 15% dextrose unless otherwise indicated. Flasks were closed with Kimble PM Caps (Rockwood, TN, USA). All incubations were at 30 °C, aerobic incubations were shaken at 100 rpm while fermentative incubations were stationary. Unless otherwise indicated inoculum was 6 ml of late log phase culture grown in the same media used in the study but without furfural. 2.2. Microorganisms Z. mobilis strain ATCCÒ 31821™ (Rhee et al., 1984) was obtained from the American Type Culture Collection. New bacterial strains that could tolerate high levels of furfural were isolated from
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various sources (compost or decaying railroad ties) as follows. Source material was diluted 1:1 (weight:volume) into sterile saline and streaked onto AmPSY agar plates supplemented with 6 g L1 furfural and 0.1 g L1 cycloheximide and incubated until colonies developed (4 days). Individual colonies were selected and tested for their ability to degrade furfural (data not shown). Based on this initial screening, three isolates (WCF2 and WCF3 isolated from compost, RR isolated from a decaying railroad tie) were selected for use in this study. All cultures were stored at 4 °C in AmPSY broth media supplemented with 3 g L1 dextrose. 2.3. Identification of strain RR, strain WCF2, and WCF3 The three newly isolated bacterial strains were identified as L. mesenteroides strains RR and WCF2 and L. pseudomesenteroides strain WCF3 based on sequencing of the 16S rRNA gene. Briefly, genomic DNA was isolated from pure cultures and 10 ng was amplified with the 27F and 1492R (Lane, 1991) primers. The resultant amplicon was cloned using the TOPO TA cloning kit for sequencing (Invitrogen, Carlsbad, CA) and sequenced in both directions using Big Dye cycle sequencing chemistry (Applied Biosystems, Carlsbad, CA). For the construction of a UPGMA tree (Fig. 1), sequences of related type strains were extracted from GenBank, aligned with ClustalW, and a UPGMA tree constructed using the MEGA 5.0 computer program (http://www.megasoft.net/) (Tamura et al., 2011). The 16S rRNA gene sequence for the WCF3 strain was deposited as accession KF698922 into the GenBank database and a culture of L. pseudomesenteroides strain WCF3 was deposited in the US Department of Agriculture, Agricultural Research Service (NRRL) Culture Collection as NRRL B-59999. 2.4. Studies The first study, a preliminary study, investigated the influence furfural had on Z. mobilis cell growth and ethanol production. The study consisted of two treatment groups and a control group, all incubated under fermentative conditions. The control group received no furfural and the two treatments groups received 1.9 and 4.3 g L1 furfural. Samples of the culture fluid were collected at the intervals indicated and assayed (see below) for growth and ethanol production. A second study, consisting of four treatment groups incubated aerobically, investigated the influence 0, 2.2, 3.5 and 4.5 g L1 amounts of furfural had on growth and furfural degradation by the three Leuconostoc strains RR, WCF2 and WCF3. Samples of the culture fluid were collected at the intervals indicated and assayed for growth and furfural content. Study three, an aerobic study, investigated the rate of furfural degradation by the three Leuconostoc strains and the principal furfural degradation product or products produced during the incubation. Only one level of furfural, 5.2 g L1 was evaluated; other conditions were as described above for study #2. Samples were collected and assayed by liquid chromatography (see below) for furfural and possible furfural degradation products. For this study, the 6 ml late log inoculum was concentrated 10-fold via centrifugation at 1620g for 15 min. The fourth and final study investigated the influence of Leuconostoc strains RR, WCF2 and WCF3 pre-treatments on the growth and ethanolic fermentation of Z. mobilis. Stage 1 incubations, the pre-treatment, were aerobic for 4 days. Stage 1 flasks were inoculated with 6 mls of late log phase cultures of the RR, WCF2 or WCF3 strains grown in HM/RSM base media supplemented with 3% dextrose; controls received 6 ml of uninoculated HM/RSM base media supplemented dextrose. Experimental media for Stage 1 contained 15% dextrose and furfural concentrations from 1.9 to 5.2 mM. For Stage 2 incubations, the ethanolic fermentation stage, the flasks
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WCF2 53 gi|116096021|L. mesenteroides subsp. mesenteroides ATCC8293T 51
gi|631252056|L. mesenteroides subsp. dextranicum JCM9700T gb|CP000414.1|:22669-24217 L. mesenteroides subsp. mesenteroides ATCC8293T
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R1 60
gi|11602803|L. mesenteroides subsp. cremoris NCFB543T gi|175133|L.mesenteroides DSM20343T
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WCF3 58 50
gi|317016875|L. pseudomesenteroides KCTC3652T gi|6683524|L. pseudomesenteroides NRIC1777T gi|221675373|L. gasicomitatum LMG18811T gi|209413803|L. palmae TMW2.694T
0.012
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Fig. 1. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 0.04082069 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. Evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1402 positions in the final dataset.
from Stage 1, after 4 days of pre-treatment, were inoculated with cells from a 3 day old culture of Z. mobilis. The Z. mobilis inoculum cells in 6 ml of RM/RSM broth supplemented with dextrose were pelleted via centrifugation at 1620g for 15 min and the pelleted cells resuspended in 6 mls of culture fluid from one of the Stage 1 pre-treatment cultures. The culture fluid plus Z. mobilis cells were added back to the original culture. Stage 2 incubations were for an additional 11 days with no shaking. Samples were collected at intervals and assayed for furfural, cell growth, dextrose content and ethanol content. 2.5. Chemicals Furfural (99% pure) was purchased from Sigma–Aldrich (St. Louis, MO); ethyl alcohol (200 proof) was obtained from Aaper Alcohol and Chemical Co. (Shelbyville, KY), and dextrose (ACS grade) was purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals were reagent grade. 2.6. Analysis Samples of the cultures (1 ml) were collected at the indicated intervals, diluted with DI water (1:9) and optical density (OD) measured at a wavelength of 660 nm. Next the diluted samples were centrifuged through Ultrafree™ 0.22 lm centrifuge filters (Millipore, Bedford, MA) spun at 10,000g for 5 min and the filtrates analyzed for dextrose, ethanol and furfural content. Furfural was estimated via a high pressure liquid chromatography (HPLC) equipped with a C-18 column and a photodiode array detector as described earlier (Hunter et al., 2012). Samples for ethanol and dextrose analysis were diluted with additional water 1:9 (v:v). Dextrose and ethanol were measured via a HPLC (LC10, Shimadzu Scientific Instruments, Columbia, MD) equipped with an AminexÒ HPX-87P 300 by 7.8 mm column (Bio-Rad Labs, Richmond, CA), a refractive index detector (RID-10A, Shimadzu) and an evaporative light scattering detector (ELSD-LTII, Shimadzu) operated at 60 °C and 360 kPa nitrogen. Injection volume was 20 ll, column temperature was 85 °C, and the mobile phase was water (Millipore MilliQ, Millipore Corp., Bedford, MA) pumped at 0.60 ml min1. Alternatively, ethanol was also measured via a gas chromatograph (GC17, Shimadzu) equipped with an Alltech Econocap ECwax column (Grace, Deerfield, IL) operated at 105 °C and with a flame ionization detector.
2.7. Statistics All controls and treatment groups consisted of three independent replicates. All data are presented as mean and standard error of the mean (SEM). For each timepoint (day) of interest, statistical significance between treatments was computed using a one-way ANOVA with the InstatÒ computer program (GraphPad Software Inc., San Diego, CA), post hoc tests (Bonferroni-corrected) were considered statistically significant for p-values below 0.05. Ethanol production rates (g L1 h1) were calculated between consecutive timepoints (e.g., Day4–Day2, Day7–Day4) using the mean treatment values (n = 3), and the highest value for each treatment, regardless of time period, is reported as the maximum ethanol production rate. 3. Results and discussion 3.1. Influence of furfural on Z. mobilis – a preliminary study Furfural, as has been shown previously (Chandel et al., 2013), had a detrimental influence on Z. mobilis growth. In this study, growth on day 1 was significantly reduced by 35% and 84% for the 1.9 g L1 and 4.3 g L1 furfural treatments, respectively (Fig. 2A). Interestingly, total ethanol production was not reduced by the two treatments though the rate at which it was produced was slowed. For example, the amount of ethanol on day 1 was significantly reduced by 33% and 92% for the 1.9 g L1 and 4.3 g L1 furfural treatments, respectively; however, by day 2 (1.9 g L1 furfural) and 7 (4.3 g L1 furfural), the total amount of ethanol present was equal to the control levels. The results show that furfural, at these levels, interfered with the Z. mobilis growth and the rate of ethanol production, but not the total amount of ethanol that could be produced. 3.2. Furfural degradation by the L. mesenteroides and pseudomesenteroides strains A second study examined the ability of the RR, WCF2 and WCF3 strains to grow in the presence of furfural as well as the ability of these L. mesenteroides and pseudomesenteroides strains to degrade furfural. The furfural treatments slowed the growth rates of all three strains, but the amounts of growth inhibition were not large (Fig. 3A–C). On day 2, the RR strain’s growth was inhibited 11–20%
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Growth (A660)
These studies showed that all three L. mesenteroides and L. pseudomesenteroides strains grew well in the presence of furfural and that all were capable of removing large amounts of furfural from the fermentation broths.
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3.3. Conversion of furfural to furfural-OH by the L. mesenteroides and L. pseudomesenteroides strains
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A third study looked at the break down products that accumulate during the degradation of furfural by the L. mesenteroides and L. pseudomesenteroides strains. Many different bacteria transform or degrade furfural and a number of different pathways are used by these bacteria. One of the simplest transformations involves the reduction of the aldehyde group to an alcohol group (Wierckx et al., 2011) yielding furfuryl alcohol (furfuryl-OH). This is a desirable transformation as the aldehyde group is more toxic than is the alcohol group. HPLC analysis showed that 89 ± 1.2% of the furfural that was lost from the incubations accumulated as furfural-OH and that furfural disappeared from the incubations at a maximum rate of 2.7 g L1 day1 (Fig. 4). Of the three strains evaluated, the WCF3 strain performed best in this study though the differences between strains were small. The formation of furfural-OH in the fermentation broth, though yields are low, might present a market opportunity. Both furfural and furfural-OH have value as industrial chemicals. Furfural-OH is used as a flavoring agent and as a fragrance; it is also used in the formation of thermosetting resins, in the production of tetrahydrofurfuryl alcohol, a chemical used as an adjuvant in herbicide formulations, and as a nonhazardous solvent (Adams et al., 1997; Oliveira et al., 2008; Song et al., 2007). Furfuryl-OH has a 2014 market price of about $1200–3000 metric ton1 in bulk while furfural has a market price of $1000–1900 metric ton1 in bulk according to Alibaba.com, an online marketplace for international trade. 3.4. Use of the L. mesenteroides and pseudomesenteroides strains as a biological pre-treatment to remove furfural A forth study evaluated the use of the L. mesenteroides and L. pseudomesenteroides strains as a method for removing furfural from the fermentation broth before the start of a Z. mobilis ethanolic fermentation. For this study furfural treatment levels of 1.9, 3.0, 4.2, and 5.2 g L1 were used with each of the three L.
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by the three furfural treatments, but by day 4 these cultures had recovered and there were no significant differences between treatments (Fig. 3A). A somewhat similar pattern (Fig. 3B) was seen with the WCF2 strain where treatment inhibitions of 18–23% were observed on day 2, but by day 4 the treatment inhibitions had declined to 0–10%. WCF3 growth, which was less than that observed with the other two strains, followed the same pattern (Fig. 3C), with day 2 growth being inhibited 15–24% by the treatments and day 4 growth inhibited by 12–18%. With all three strains, furfural levels in the culture fluid decreased during the incubations. The largest and most rapid decreases were observed with the RR strain where, by day 4, furfural levels ranged from 0.0 to 0.91 g L1 (Fig. 3D). With the WCF2 and WCF3 strains furfural levels decreased to 0.0 to 1.4 and 0.0 to 1.6 g L1, respectively, by day 4 (Fig. 3E and F).
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Fig. 3. The ability of L. mesenteroides and pseudomesenteroides strains to grow in the presence of furfural (A, B, and C) and their ability to degrade furfural (D, E, and F).
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RR Maximum degradation rate = 2.7 ± 0.07 g L-1 Day-1
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Fig. 4. The conversion of furfural to furfural-OH by L. mesenteroides and pseudomesenteroides strains.
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mesenteroides and L. pseudomesenteroides strains. During the Stage 1 pre-treatment the three strains RR, WCF2 and WCF3 removed >99% of the 1.9 g L1 furfural initially present, 86–99% of the 3.0 g L1 furfural initially present, 81–90% of the 4.2 g L1 furfural initially present, and 62–66% of the 5.2 g L1 furfural initially present (Fig. 5A–D); thereby, significantly reducing the amount of furfural present at the start of the ethanolic fermentation stage. The uninoculated negative control confirmed that the furfural was not lost due to volatilization, degradation, etc. The negative control data also showed that Z. mobilis alone was able to degrade furfural during Stage 2 (ethanolic fermentation stage); and by day 2, Z. mobilis had degraded 95% of the furfural from the 1.9, 3.0 and 4.2 g L1 furfural treatments (Fig. 5A–C), but only 86% of the furfural from the 5.2 g L1 furfural treatment level (Fig. 5D). At the end of the study (day 11), the Z. mobilis cells had removed only 85% of the furfural (i.e., 0.73 g L1 remaining); whereas, all three Leuconostoc strains had removed 100%.
increased Z. mobilis growth on day 2 by 85% and on day 4 by 65%, almost reversing the inhibition caused by furfural on these days (Fig. 5E). In contrast, the WCF2 and RR strains only increased Z. mobilis growth on these days by averages of 45% and 6%, respectively. At the higher amounts of furfural the differences between the L. mesenteroides and L. pseudomesenteroides strains became less noticeable, and in no instance did the pre-treatment restore full Z. mobilis growth (e.g., similar to that of the positive controls) . 3.6. Impact of furfural and the pretreatments on Z. mobilis dextrose uptake Dextrose utilization by all three Leuconostoc strains was very low during the Stage 1 pre-treatments, but appeared to increase with increasing furfural concentrations. For example, the average dextrose consumption by the three Leuconostoc strains was 3.8%, 2.1%, 5.1%, and 16.4% for the 1.9, 2.0, 4.2, and 5.9 g L1 treatments, respectively (Fig. 6A–D). The Stage 1 pre-treatments had a clear impact on dextrose uptake during the Stage 2 ethanolic fermentations. Furfural had a progressive and negative impact on dextrose uptake by Z. mobilis when no L. mesenteroides and L. pseudomesenteroides pre-treatments were employed (Fig. 6A–D). For the 1.9 g L1 furfural treatment, no significant differences in Z. mobilis dextrose consumption were observed, with dextrose consumption nearly complete (> 90%) by day 4 for all of the treatments (Fig. 6A). However, for the 3.0 and 4.2 g L1 treatments (Fig. 6B and C) dextrose consumption was nearly complete (>90% consumed) by day 4 for all three Leuconostoc pre-treatments, but not until day 7 for the negative control. The 5.2 g L1 treatment essentially prevented dextrose uptake in the negative control (Fig. 6D), which could be restored by pre-treatment with all three Leuconostoc strains; however, dextrose consumption was still delayed (i.e., >90% consumption at day 7).
3.5. Impact of furfural and the L. mesenteroides and pseudomesenteroides pre-treatment on Z. mobilis cell growth As was seen in the preliminary study (Fig. 2A) a comparison of data from the positive control (no furfural) and negative control (furfural but no L. mesenteroides or L. pseudomesenteroides pretreatment) shows that the presence of furfural in the fermentation media reduced the growth of Z. mobilis cells (Fig. 5E–H). The growth inhibition of Z. mobilis caused by the furfural treatments was reduced by the Stage 1 pre-treatments, with all three of the L. mesenteroides and L. pseudomesenteroides strains improving the growth of Z. mobilis despite the high level of furfural in the original media. The WCF3 strain preformed best in most of these studies followed by the WCF2 strain and then by the RR strain. At the 1.9 g L1 furfural level the WCF3 Stage 1 pre-treatment
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Fig. 5. Furfural concentration and cell growth during the two stage incubations. The first portion (Stage 1) of the incubation was an aerobic 4 day pre-treatment with no inoculum, the control, or with one of three L. mesenteroides or pseudomesenteroides strains as the inoculum. The second portion (Stage 2) of the incubation, lasting from day 0 to day 11, was an ethanolic fermentation. All incubation flasks were inoculated with Z. mobilis on day 0.
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Fig. 6. Dextrose and ethanol concentration during the two stage incubations. The Stage 1 incubation was an aerobic 4 day pre-fermentation incubation with no inoculum or with one of three L. mesenteroides or pseudomesenteroides strains as the inoculum. The Stage 2 incubation, lasting from day 0 to day 11, was a fermentative incubation. All incubation flasks were inoculated with Z. mobilis on day 0.
Most importantly, during the pre-treatment all three Leuconostoc strains did not appear to consume large amounts of dextrose. The average loss of dextrose, across all treatments, was only about 5% during the 4 day pre-treatment incubation. 3.7. Impact of furfural and the pretreatments on ethanol production As was the case in the preliminary study (Fig. 2B), furfural had a clear negative impact on ethanol production by Z. mobilis; however, the Stage 1 pre-treatments with the L. mesenteroides and L. pseudomesenteroides strains were able to partially alleviate the inhibition caused by furfural. Without the pre-treatment, positive controls vs negative controls (Fig. 6E–H), furfural had a generally progressive and negative impact on ethanol production during the Stage 2 fermentations. At the 1.9 g L1 level furfural delayed the onset of ethanol production by about a day and reduced the maximum rate of ethanol production by about 42% (Fig. 6E), from 1.34 to 0.76 g L1 h1; while the 3.0 and 4.2 g L1 treatments (Fig. 6F and G) delayed the onset of ethanol production by 2 and 4 days, respectively, and retarded production by 70%. The highest furfural treatment level, 5.2 g L1 essentially stopped ethanolic fermentation (Fig. 6H). The L. mesenteroides and L. pseudomesenteroides pretreatments reduced the inhibition caused by furfural and increased the rate of ethanol production relative to the negative controls. Pretreatments with the RR, WCF2 and WCF3 strains increased the maximum ethanol production rates in the 1.9 g L1 furfural treatment group to 0.98, 1.60, and 2.19 g L1 h1, respectively. In the 3.0 g L1 furfural treatment group the negative control rate was 0.55 g L1 h1 and the fermentations that were pretreated with RR, WCF2 and WCF3 in Stage 1 had maximum ethanol production rates of 1.18, 1.43, and 1.86 g L1 h1, respectively. In the 4.2 g L1 furfural treatment group the negative control rate was 0.67 g L1 h1 and the fermentations that were pretreated with RR, WCF2
and WCF3 in Stage 1 had production rates of 0.92, 0.77, and 1.26 g L1 h1, respectively. In the 5.2 g L1 furfural treatment group the negative control rate was 0.05 g L1 h1 and the fermentations that were pretreated with RR, WCF2 and WCF3 in Stage 1 formed ethanol at maximum rates of 0.72, 0.66 and 0.56 g L1 h1, respectively. 4. Conclusions Furfural greatly reduced or prevented growth and ethanol production by Z. mobilis cells but not the growth of Leuconostoc cells. Moreover, Leuconostoc cells degraded furfural to the less toxic furfural-OH. A biological pre-treatment of a model fermentation broth with Leuconostoc cells reduced the furfural in the broth improving Z. mobilis growth and, most importantly, ethanol production. The Leuconostoc strains did not consume large amounts of dextrose during the pre-treatment and did not significantly reduce the total amount of ethanol formed, relative to non-furfural containing controls. Additionally, the furfural-OH formed by the Leuconostoc cells has commercial value. Acknowledgements The authors thank Robin Montenieri and Joshua Padilla for their skilled technical support. Manufacturer and product brand names are given for the reader’s convenience and do not reflect endorsement by the US government. This article was the work of US government employees engaged in their official duties and is exempt from copyright. References Adams, T.B., Doull, J., Goodman, J.I., Munro, I.C., Newberne, P., Portoghese, P.S., Smith, R.L., Wagner, B.M., Weil, C.S., Woods, L.A., Ford, R.A., 1997. The FEMA
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