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Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate

Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium capable of utilizing xylose for the fermentation production of butyrate. Hot water extraction of hardwood lingocellulose is an efficient method of producing xylose where autohydrolysis of xylan is catalysed by acetate originating from acetyl groups present in hemicellulose. The presence of acetic acid in the hydrolysate might have a severe impact on the subsequent fermentations. In this study the fermentation kinetics of C. tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate equivalents were studied. Analysis of xylose batch fermentations found that even in the presence of high levels of acetate, acetate adapted strains had similar fermentation kinetics as the parental strain cultivated without acetate. The parental strain exposed to acetate at inhibitory conditions demonstrated a pronounced lag phase (over 100 hours) in growth and butyrate production as compared to the adapted strain (25 hour lag) or non-inhibited controls (0 lag). Additional insight into the metabolic pathway of xylose consumption was gained by determining the specific activity of the acetate kinase (AK) enzyme in adapted versus control batches. AK activity was reduced by 63% in the presence of inhibitory levels of acetate, whether or not the culture had been adapted. Keywords: Clostridium tyrobutyricum, Butyrate, Xylose fermentation, Hemicellulose utilization, Acetate inhibition Introduction microbial growth (Helmerius, et al. 2010). When used in Butyric acid is approved by the Food and Drug Adminis- fermentation media, the inhibitory acetate generates a tration (US) as a flavor enhancer and several flavor esters long lag period before log phase growth and butyric acid used in the food industry are derived from butyric acid. production (Jaros, et al. 2012). Previous work has shown There is a well established market for all-natural foods, that addition of 17.6 g/L and 26.3 g/L acetate in the media where the components are not synthetically derived from generates a lag phase of 45 and 118 hours respectively petro-chemicals as well as a strong consumer bias against while un-inhibited controls begin fermentation and subse- using genetically modified organisms (GMOs) in food quently production almost immediately upon inoculation production. Due to this, butyric acid fermented from bio- (Jaros, et al. 2012). mass by wild type anaerobic bacteria can be developed as Multiple Clostridial strains have been classically selected a saleable commodity. for increased tolerance to both butanol and ethanol which Un-utilized hemicellulose streams from the pulp and successfully lead to higher solvent yields and higher over- paper industry can potentially, after hydrolysis, provide all productivity (Lin and Blaschek 1983; Herrero and a low-cost source of xylose feedstock for organic acid Gomez 1980). Due to the toxicity of these compounds, fermentation. Hardwood xylan is extensively acetylated, each step of the selection requires a short unchallenged i.e. up to seven acetyl groups per ten xylose units which incubation period, an exigency removed when challenging facilitate xylose release by autohydrolysis (Teleman, et al. the organism with acetate. 2002). The resulting hemicellulose hydrolysate contains For organic acid production, non-solventogenic Clostridia levels of acetate of up to 40 g/L acetic acid, inhibitory to such a C. tyrobutyricum are used in fermentation processes where none of the typical toxic by-products such as butanol and ethanol are produced. C. tyrobutyricum cultures have * Correspondence: Ulrika.Rova@ltu.se Luleå University of Technology, Luleå SE-971 87, Sweden been selectively adapted to tolerate the presence of Michigan State University, East Lansing, MI 48824, USA © 2013 Jaros et al.; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jaros et al. SpringerPlus 2013, 2:47 Page 2 of 8 http://www.springerplus.com/content/2/1/47 inhibitory organic acids in order to increase acid product fermenting wild type cultures (Jaros, et al. 2012). This sim- yields (Zhu and Yang 2003). Despite their success, these ple means of directing carbon flux towards butyric acid selections have been performed on immobilized production is an added benefit of working with high acetate C. tyrobutyricum cultures in fibrous-bed bioreactors media and is especially important in light of evidence that requiring a 3 day cell growth period followed by a 36 to such levels of acetate are present in potential xylose feed- 48 hour cell immobilization period in order for a conti- stock streams (Helmerius, et al. 2010; Jaros, et al. 2012). nuous feed fermentation to begin (Zhu and Yang 2003). C. tyrobutyricum batch fermentations under high acetate Such a process allows for the eventual in-line adaptation challenged conditions perform better with xylose as a car- of a C. tyrobutyricum culture to inhibitory acid products bon source than glucose. Fermentations with 26.3 g/L ini- while simple adaptation techniques produce a tole- tial acetate generated 32.6 g/L butyric acid on xylose, while rant culture ready to inoculate immediately into the comparable batch with glucose feed produced 22.3 g/L batch fermentation. (Jaros, et al. 2012). Similar results were received with all Through our work we have detected that C. tyrobutyricum initial acetate concentrations (0, 4.4, 8.8, 17.6 g/L). How- demonstrates diauxic growth, the phenomena of a meta- ever batch fermentations utilizing high acetate (26.3 g/L bolic shift occurring in the middle of the growth cycle initial acetate) xylose synthetic media resulted in an when the two carbon sources glucose and xylose are pre- extended lag phase of 118 hours, lowering productivity sent (data not shown). The presence of a more utilizable (Jaros, et al. 2012). The extended lag phase generated by carbon source, in this case, glucose, prevents activation acetate is economically detrimental for batch fermentation of the metabolic machinery required for the cells to of butyrate as it leads to a long period of reactor inactivity consume the secondary substrate, xylose. Fortunately, and potential exposure to microbial contamination. On the C. tyrobutyricum readily consumes xylose if the culture other hand, after lag phase the 26.3 g/L initial acetate has been pre-conditioned to xylose metabolism and no challenged batch obtained a similar biomass concentration other sugar sources are available. as the lower acetate and control batches and surpassed Anaerobic, butyrate producing bacteria such as Clostridia them in final butyrate yield (Jaros, et al. 2012). The focus metabolize glucose to pyruvate through the Embden- of this work is to adapt a strain of C. tyrobutyricum to in- Meyerhof-Parnas (EMP) pathway and concomitantly creased acetate tolerance, thus decreasing the extended lag generate acetate, butyrate, H and CO as major meta- phase while maintaining the acetate re-utilization meta- 2 2 bolic end-products (Zhang, et al. 2009). Xylose is speci- bolic mechanism to deliver increased yields of butyric acid. fically catabolised in the Hexose Monophosphate Pathway As hardwood derived hemicellulose hydrolysate feedstock to pyruvate which is enzymatically co-oxidized with cellu- gives rise to high levels of both xylose and acetate, a xylose lar coenzyme-A to acetyl coenzyme A (Zhu and Yang consuming strain capable of overcoming the acetate in- 2004; Madigan, et al. 2009). Acetyl-CoA is the branch- duced lag and yet re-utilizing acetate to generate even point node of the acetate and butyrate end-product path- more butyric acid would be of commercial value. ways where the enzymes phosphotransacetylase (PTA) and acetate kinase (AK) are responsible for the metabo- Methods lism of acetyl-CoA to acetate if the branch-point does not Microorganism and adaptation follow the butyrate pathway (Zhu and Yang 2004). In A lyophilized stock culture of C. tyrobutyricum (ATCC attempts to force the carbon flux from the acetate to bu- 25755) was re-hydrated under sterile anaerobic conditions tyrate metabolic branch in C. tyrobutyricum, mutants have in Reinforced Clostridial Media (RCM; Difco). Once the been developed with inactivation’sin the pta and ack culture entered log phase, when the optical density (OD) genes coding for PTA and AK respectively (Zhu, et al. at 600 nm was approximately 2.0, transfers were made 2005; Liu, et al. 2006). Fermentations with the mutants to glycerol stock vials (CRYOBANK ) and the culture yielded more butyric acid compared to wild type was maintained at −70°C. C. tyrobutyricum was classically C. tyrobutyricum, but both mutant strains demonstrated adapted to 26.3 g/L inhibitory acetate equivalents by serially significantly slower growth kinetics than wild type and in passaging log phase cultures into serum bottles with RCM both cases resulted in higher final acetic acid concentrations containing subsequently higher concentrations of sodium with increased acid tolerance (Zhu, et al. 2005; Liu, et al. acetate (starting at 0 g/L then, 6 g/L, 12 g/L, 24 g/L and 2006). These results exhibit a common issue of genetic 36 g/L sequentially) at each passage. As the molar mass of engineering in that GMO’s are typically less robust than sodium acetate is 82.03 g/mol, these concentrations corres- wild type (slower growth) and the complexity of most pond with 0 g/L, 4.4 g/L, 8.8 g/L, 17.6 g/L, and 26.3 g/L metabolic pathways allows for the re-routing of acetic acid equivalents respectively. inactivated processes due to homeostasis. The presence of The adaptation was performed on two sets of C. 17.6 g/L to 26.3 g/L initial acetate in the media has the tyrobutyricum cultures, each culture solely conditioned similar effect of lowering acetate production in xylose to consuming either xylose or glucose so that the actual Jaros et al. SpringerPlus 2013, 2:47 Page 3 of 8 http://www.springerplus.com/content/2/1/47 batch fermentations could be performed without a lag fermentation. Sodium acetate (0 – 36 g/L) was added to phase due to an altered sugar source. The glucose con- the initial media prior inoculation for studies assessing ditioned culture was maintained with RCM from Difco acetate inhibition. Fermentations without acetate are re- with the appropriate additions of acetate equivalents in ferred to as controls. Samples (10 mL) were withdrawn at the form of sodium acetate. The xylose conditioned cul- regular intervals for analytical measurements. Data ture bottles also received the appropriate amount of ace- presented in the tables and figures of this study are the tate equivalent from a media consisting of: 10 g peptone results of single batch fermentations while an analysis in- (Fisher), 10 g beef extract (Teknova), 3 g yeast extract volving duplicate and triplicate fermentations is given in (Bacto), 5 g sodium chloride (J.T. Baker), 0.5g L-cysteine the discussion where stated. (Sigma-Aldrich), 3g sodium acetate anhydrous (J.T. Baker), 0.5 g agar (Bacto) and 900 mL distilled water. For the Analytical methods xylose feed, 5 g of xylose (Acros) in 10 mL distilled water, Organic acids and residual sugar were analyzed by HPLC separately autoclaved at 121°C for 20 min was added to the (LC-20AT dual pump and 10A RI detector, Shimadzu) culture media. Prior to autoclaving all serum bottles were equipped with an ion exchange column (Aminex HPX- sparged with nitrogen to maintain an anaerobic atmos- 87H, 9 um, 7.8 mm x 300 mm, Bio-Rad) and a cation-H phere. Each serum bottle contained a total volume of 100 guard column (Micro-guard, 30 mm × 4.6 mm) using 50 mL RCM (initial pH 6.5) with 5 mL from the previous mM sulfuric acid as a mobile phase. The flow rate of the stage used to inoculate the next higher acetate stage. Du- mobile phase was maintained at 1 mL/min during analysis ring adaptation, serum bottles were incubated at 36°C in with 20 μL of sample injected into the system with an an incubator-shaker (New Brunswick Scientific Innova 40) auto-injector (SIL-20AHT, Shimadzu) with the column with shaking at 80 rpm. and guard maintained at 65°C in a column oven (CT0- The cultures required 24 hours to adapt and reach log 20A, Shimadzu). Prior to analyses, samples were centri- phase growth before passaging to the next level of selec- fuged at 10 000 rpm for 5 min in a micro-centrifuge tion with the exception of the last transfer of the 17.6 g/L (Microfuge 18, Beckman Coulter). Data for each sample acetate adapted cultures to the final 26.3 g/L. Glucose was acquired with Shimadzu EZ Start 7.4 SP1 chromatog- conditioned cultures required 48 hours to reach log phase raphy software using standards for glucose, xylose, butyr- when challenged with 26.3 g/L acetic acid and xylose ate, acetate and lactate. conditioned required 96 hours of incubation to reach log phase. C. tyrobutyricum inoculum for each batch fermenta- Dry cellular weight determination tion were pre-conditioned to the correct sugar substrate Cell growth was monitored during fermentation by meas- in the inoculation media prior the batch fermentation by uring the optical density at 600 nm. The biomass from 40 anaerobically inoculating 50 mL Screw Cap Corning mL cell suspension, removed in triplicate, was dried in an tubes containing 35 mL sterile glucose or xylose based 80°C drier for 48 hours and the dry cell weight (DCW, g/L) RCM with 5 mL of the stock culture. The inoculated tubes determined. The optical densities were then converted to were cultivated under anaerobic conditions at 36°C, 80 dry cell weight using the following equation: DCW = 0.38 rpm, until log phase, approximately when OD had (OD ). This optical density to dry cellular weight conver- reached a value of 2. 600 sion formula was determined for the specific organism and media used in this study. Fermentations One liter batch fermentations were conducted in New Brunswick Bioflo 310 2.5 L working volume reactors under anaerobic conditions at 36°C. For each batch, 950 Specific Growth Rate (μ ) net mL media of the following composition was used; 6 g/L DCW was used to determine the specific growth rate as yeast extract, 5 ppm FeSO 7H O, and 200 mL xylose described by Shuler et al. (Shuler and Kargi 2002). The 4 2 or glucose at 300 g/L sterilized separately. Anaerobiosis DCW data points from the logarithmic growth phase was reached by sparging the vessel with nitrogen prior were plotted on a semi-log graph to locate the period to inoculation. The batches were inoculated with 50 mL during that phase in which the culture experienced the log phase C. tyrobutyricum cultures. The nitrogen spar- fastest growth. These points were then used in the ging was maintained until logarithmic growth in the ves- following equation: μ (1/h) = (ln(DCW /DCW ))/ net x 0 sel was observed. (Time -Time ), where DCW was measured in g/L and x 0 Agitation was kept at 250 rpm and in order to maintain time in hours. DCW is the last point during the fasted the C. tyrobutyricum cultures in acidogenic production, logarithmic growth period and DCW is the first point. pH 6.0 was sustained with 5 M NaOH throughout the Time and Time are described similarly. x 0 Jaros et al. SpringerPlus 2013, 2:47 Page 4 of 8 http://www.springerplus.com/content/2/1/47 Acetate kinase assay consumption almost immediately with butyric acid pro- Bacterial cells from xylose conditioned batches at log phase duction beginning 15 hours later (Figure 1a and 1b). growth were chilled on ice and centrifuged at room The same culture inoculated into xylose-minimal media temperature at 5,000 rpm for 5 min and washed in 25 mM containing 26.3 g/L acetate equivalents required over Tris–HCl, pH 7.4 in order to remove acetate from the 100 hours to acclimate to the acetate despite both medium. After a second centrifugation the cell pellet was fermentations operating under the same conditions. The resuspended in 25 mM Tris–HCl, pH 7.4 and sonicated extended period of minimal metabolism and productivity three intervals at 30 khz for 60 seconds, while on ice, to is due to the acetate causing a delay in log phase cellular lyse the cell wall. The supernatant was used for acetate ac- growth (Figure 1c). Once the C. tyrobutyricum culture tivity studies using a method (Rose 1955) where the con- had adapted to the 26.3 g/L acetate media the culture version of acetate to acyl phosphates by acetate kinase is performed like the control, resulting in complete xylose coupled to the formation of a ferric-hydroxamate complex utilisation and production of over 25 g/L of butyric acid detectable by UV–visat540 nm.In summary,the enzyme and similar levels of cell mass. activity was measured at 29°C using UV/VIS spectroscopy The acetate adapted culture maintained tolerance to where the absorbance of a 4 mL reaction mixture at the 26.3 g/L acetate in the media and after a 22 hour lag 540 nm and the ferric-hydroxamate complex molar extinc- in xylose consumption following inoculation, subse- -1 -1 tion coefficient of 0.169 mM cm was used to calculate quently began producing butyric acid (Figure 1a and 1b). the enzyme activity (Zhu and Yang 2003; Zhu, et al. 2005). The acetate tolerant culture running under acetate in- Acetate kinase activity was standardized to the total pro- hibition conditions performed similar to the control fer- tein content of each sample, determined separately by mentation in that the xylose was fully utilized in 175 Bradford (Bio-rad protein assay) using bovine serum albu- hours from inoculation and produced 28 g/L butyric min. One unit of acetate kinase is defined as the amount of acid compared to the controls production of 25.8 g/L enzyme producing 1 μmol of hydroxamic acid per minute butyric acid. Despite the increased product yield, the net at 29°C and the specific activity calculated as units of specific growth rate (μ ) of the acetate tolerant culture net activity/mg cellular protein.The resultsreportedhere was reduced by 28.7% compared to the control. The are averages of enzyme assays run in triplicate. specific growth rate of the control fermentation was 0.093 1/h while the acetate selected culture showed a Results log phase growth of 0.067 1/h (Table 1). This observa- Fermentation kinetics tion is not surprising as a similar yield increase The non-adapted (control) C. tyrobutyricum culture corresponding with a growth rate reduction was seen in inoculated into xylose-minimal media begins sugar genetically modified C. tyrobutyricum where the pta No external acetate with non-adapted culture 26.3 g/L external acetate with adapted culture 26.3 g/L external acetate with non- adapted culture 0100 200 300 Time (h) bc 35 3.5 30 3.0 25 2.5 20 2.0 15 1.5 10 1.0 5 0.5 0 0.0 0100 200 300 0 100 200 300 Time (h) Time (h) Figure 1 Impact of acetate on xylose consumption, butyric acid production and biomass generation. Butyric Acid equivalents Xylose (g/L) (g/L) DCW (g/L) Jaros et al. SpringerPlus 2013, 2:47 Page 5 of 8 http://www.springerplus.com/content/2/1/47 Table 1 Fermentation kinetics of C. tyrobutyricum cultures run in batch with or without selection for acetate tolerance and with or without acetate inhibition Sugar Acetate C. Lag Complete Sugar Butyrate Final concentration Specific Growth Overall 2 3 4 5 7 8 tyrobutyricum time utilization of cons Yield Rate (μ ) produc. net carbon (g/L) (h) (h) (g/L/h) (mol/mol) (g/L) (1/h) (g/L/h) Butyrate Acetate Bio- mass Glc 0 non-adapted 0 77 1.07 0.85 25.61 8.38 3.40 0.306 0.28 Glc 26.3 non-adapted 94 171 1.09 0.89 26.22 27.85 3.59 0.274 0.15 Glc 26.3 adapted 0 75 1.21 0.87 25.86 32.03 2.77 0.206 0.32 Xyl 0 non-adapted 0 166 0.56 0.74 25.80 4.24 2.72 0.093 0.16 Xyl 26.3 non-adapted 102 167 1.22 0.79 29.00 27.76 3.04 0.121 0.12 Xyl 26.3 adapted 25 174 0.60 0.81 28.92 24.46 2.30 0.067 0.17 Glucose and xylose respectively. Whether or not the inoculum had been selectively adapted to 26.3 g/L. Calculated as time until sugar consumption started. Calculated for the linear sugar consumption phase. Yield was calculated as mol butyrate per mol glucose or xylose consumed during fermentation. Calculated as DCW g/L. 7 -1 As determined by the formula μnet (h ) = (ln(DCW /DCW ))/(Time -Time ). x 0 x 0 Overall productivity calculated from the start of the fermentation until the sugar source were completed. gene had been deleted (Zhu, et al. 2005; Liu, et al. non-adapted-inhibited and adapted-inhibited) generated 2006). very similar levels of butyric acid between batches (25.61, The effectiveness of selective adaptation to generate an 26.22 and 25.86 g/L respectively) (Table 1). Analogous to acetate tolerant C. tyrobutyricum culture is even more the xylose batches, the acetate inhibited non-adapted cul- evident in glucose consuming fermentations. The adapted ture experienced approximately 94 hours of lag phase be- inoculum under 26.3 g/L acetate conditions experienced fore beginning to consume glucose, produce butyric acid no lag in growth and tracked almost exactly with the unin- or generate DCW biomass (Figure 2a-c, Table 1). Acetate hibited control in terms of glucose consumption and adaptation allows the culture to overcome inhibition butyric acid production (Figure 2a and 2b). Unlike the caused by 26.3 g/L acetate and the 94 hours of lag phase. xylose batches, the glucose consuming cultures (control, A net production of acetate occurred in the glucose No external acetate 60 with non-adapted culture 26.3 g/L external acetate with 30 adapted culture 20 26.3 g/L external acetate with non- adapted culture 0100 200 300 Time (h) bc 30 4 3.5 2.5 15 2 1.5 0.5 0 0 0100 200 300 0 100 200 300 Time (h) Time (h) Figure 2 Impact of acetate on glucose consumption, butyric acid production and biomass generation. Butyric Acid equivalents Glucose (g/L) (g/L) DCW (g/L) Jaros et al. SpringerPlus 2013, 2:47 Page 6 of 8 http://www.springerplus.com/content/2/1/47 consuming acetate adapted batch demonstrating the shown), compared to 94 hours for the non-adapted higher cellular energy made available from glucose con- strain. In contrast, there was a complete reversion of sumption as compared to that of xylose. The xylose con- the acetate adapted strain during xylose fermentation suming acetate adapted batch activated the Clostridial usinganinoculumfromcryogenic storage. Further acetate re-utilization pathway resulting in an overall con- characterization of strain stability and the molecular sumption of acetate rather than production. This activa- mechanisms resulting in increased tolerance for acetate tion was likely necessitated by the lower amount of energy is needed to identify target enzyme pathways or individ- from xylose metabolism. ual genes important for the desired phenotype. The Similar to the xylose batches, the acetate tolerant culture induced tolerance of C. tyrobutyricum enables one to consuming glucose also exhibited a 32.7% reduction in spe- use adaptation as a tool to identify alteration of the cific growth rate compared with the glucose control culture organism's own enzyme systems that can be targeted for (Table 1). The glucose control batch demonstrated a 0.306 further permanent genetic modification. 1/h specific growth rate and the adapted culture dropped to 0.206 1/h during acetate inhibition (26.3 g/L). The non- Acetate kinase activity adapted culture under acetate inhibition (26.3 g/L) dropped The metabolic selectivity in C. tyrobutyricum is influenced to 0.274 1/h, only a 10.5% reduction compared to the glu- by growth stage, with exponentially growing cultures pro- cose control batch. ducing both butyric and acetic acids, while slower station- The specific growth rates of glucose consuming ary growth rates tend towards butyric acid (Michel-Savin, batches were two to three times higher than those of the et al. 1990). As such, during log phase growth of each xylose consuming C. tyrobutyricum batches (Table 1). batch, culture samples were removed and analyzed for Lowered specific growth rates are a consequence of xy- acetate kinase activity. Acetate kinase (AK) is the last en- lose consumption due to the lowered energetic value of zyme on the metabolic arm converting acetyl-CoA to xylose metabolism over glucose. With less free energy acetate, thus AK activity under particular fermentation from sugar consumption, the xylose consuming batches conditions is related to acetate production (Liu, et al. have less energy to perform cellular maintenance and 2006). Table 3 presents the specific activity in relation growth thus, in general have lower specific growth rates to cellular protein. The presence of inhibitory acetate than glucose consuming batches. (26.3 g/L) in the media reduced the AK activity to 3.15 The xylose consuming acetate-inhibited batches exhibited U/mg in both the adapted and non-adapted cultures as higher final yields of butyric acid than the control culture the control culture exhibited 8.42 U/mg (Table 3). In (Table 2). Both the acetate tolerant and non-adapted both cases of acetate inhibition, whether the culture was cultures yielded 0.48 g/g butyric acid from the initial 60 g/L acetate tolerant or not, the acetate kinase activity was xylose compared to the control cultures 0.43 g/g. Glucose reduced leading to the inhibition of metabolic acetate consuming cultures demonstrated no significant change in production (Figure 3, Table 1). butyric acid yield between the 3 batches (Table 2). The AK specific activity results correlate strongly to The selection pressure during cultivation in 26.3 g/L the production data in Figure 3, where the control cul- acetate medium with xylose or glucose resulted in a ture with the highest AK activity also generated the strain with improved butyrate production while exposed most acetic acid equivalents, 4.24 g/L. The non-adapted to high acetate concentrations during fermentation. batch with 26.3 g/L initial acetic acid equivalents and However, this phenotype was only preserved to some the lowered AK activity generated only an additional extent for the glucose fermenting acetate adapted strain. 2.65 g/L acetic acid by the time the xylose had been When this adapted strain, stored at −70°C, was used dir- completely utilized (Figure 3). The selected batch run ectly to inoculate a 26.3 g/L acetate challenged media, under the same initial acetic acid conditions performed the lag phase was increased to 42 hours (results not Table 3 The impact of the presence of acetate on enzymatic Acetate Kinase activity in C. tyrobutyricum Table 2 The effect of acetate inhibition on butyric acid fermentations yield in batch fermentations of C. tyrobutyricum with an No external 26.3 g/L 26.3 g/L initial 60 g/L glucose or xylose and run until completion acetate with external external Butyric acid yield (g/g) non-adapted acetate with acetate with culture non-adapted adapted Carbon No external 26.3 g/L external 26.3 g/L external culture culture source acetate with acetate with non- acetate with non-adapted adapted culture adapted culture Acetate Kinase 8.42 3.15 3.15 culture activity (Units/mg cellular protein) Glucose 0.43 0.44 0.43 Results reported here are averages of enzymes assays run in triplicate as Xylose 0.43 0.48 0.48 described in the methods. Jaros et al. SpringerPlus 2013, 2:47 Page 7 of 8 http://www.springerplus.com/content/2/1/47 negligible quantities of acetic acid even during the begin- ning log phase stage (Figure 3). Other than AK inhibition, another innate mechanism No external acetate with pushing the carbon flux of the Clostridial metabolism non-adapted culture towards butyrate and away from acetate is the re-uptake 26.3 g/L external acetate of acetate from the media back into the usable acetyl- with acetate adapted culture 0100 200 300 CoA pool by the CoA transferase enzyme (Michel-Savin, -1 26.3 g/L external acetate with non-adapted culture et al. 1990). This re-utilization mechanism of acetate -2 provides no energy benefits to the cell but allows for the -3 control of environmental acetate and utilizes protons in the -4 Time (h) acetate-to-butyrate conversion process (Michel-Savin, et al. Figure 3 Effect of acetate inhibition on relative acetic acid 1990). Acetate re-uptake can be exploited under the fermentation kinetics of C. tyrobutyricum xylose batches. conditions pertaining to a microbial inhibiting level of acetate present in the feed stream since the supposed con- with even higher carbon flux away from the acetate taminant in this case can potentially be used as a carbon branch as acetate re-uptake mechanisms allowed the source (Helmerius, et al. 2010; Jaros, et al. 2012). Some of culture to consume 2.47 g/L of the initial acetate from the re-assimilated acetyl-CoA enters the butyrate pathway the media (Figure 3). and thus this mechanism contributes to carbon efficiency (Canganella, et al. 2002). Acetate re-uptake occurred in Discussion the xylose consuming pre-adapted fermentation, not Acetate tolerant C. tyrobutyricum cultures consuming only is the final butyric concentration (28.92 g/L) higher xylose overcame the acetate induced lag growth phase than the control (25.8 g/L) but the initial acetate con- four times faster than the comparable non-selected centration decreases during the course of the study cultures under the same acetate inhibition conditions (Figure 1b and 3). Unfortunately, CoA transferase is also (26.3 g/L) (Figure 1a-c, Table 1). The selected culture implicated in a redundant pathway leading to acetate gen- also maintained lowered utilization of the acetate meta- eration directly from acetyl-CoA, so information concer- bolic pathway under challenged conditions (Figure 3 and ning this enzymes specific activity may not provide useful Table 3). The acetate producing metabolic pathway yields information concerning the acetate re-uptake mechanism more ATP than the butyrate pathway, so an inhibition of (Liu, et al. 2006). acetate kinase (AK) or phosphotransacetylase (PTA) leads The selective adaptation of acetate tolerant glucose to increased carbon flux towards phosphotransbutyrylase consuming cultures completely eliminated the acetate (PTB) and butyrate kinase (BK) as the butyrate pathway induced lag phase in growth under inhibitory conditions must compensate for the energy loss (Zhu and Yang 2004; (Figure 2a-c, Table 1). The higher energetic value of glu- Michel-Savin, et al. 1990). Rather than lower energy con- cose consumption over that of xylose appears to allow sumption and less biomass generated, the acetate inhibited acetate selected cultures consuming glucose to begin C. tyrobutyricum cultures generated a similar amount of fermentation immediately even under 26.3 g/L acetate biomass as the control by increasing butyrate production inhibition (Figure 2a). This is remarkable given that the to overcome the energy inefficiency (Figure 1c, 2c). Similar non-selected glucose consuming batch still required a 94 to our results, C. tyrobutyricum fermentations with genetic hour lag-phase to overcome acetate inhibition, similar to inactivation of pta also had higher butyric yields and the 102 hours seen in the xylose consuming non- inactivated (or in our case, inhibited) acetate producers still selected culture under the same conditions (Table 1). developed similar levels of biomass as controls (Figure 1c, The selective adaptation of C. tyrobutyricum for acetate tolerance is more effective for glucose consuming 2c) (Zhu, et al. 2005). Both acetate kinase and phosphotransacetylase are more cultures than xylose consumers. sensitive to product inhibition by butyrate than the The energetic differences between xylose and glucose consumption appear to also affect the final butyric acid enzymes responsible for the butyrate pathway, butyrate kinase and phosphotransbutyrylase (Zhu and Yang 2003). yields for 26.3 g/L acetate inhibited batches (data not This natural inhibition is beneficial from an industrial shown). Duplicate fermentations of 60 g/L xylose produced an average of 27.16 g/L butyric acid with a standpoint as shortly after the culture enters the exponen- tial growth phase C. tyrobutyricum stops co-producing standard deviation of (+/− 1.93) while duplicate fer- both acid products and singularly forms butyrate (Michel- mentations of 60 g/L dextrose average 24.34 g/L butyric acid (+/− 0.99), a non-significant difference. Challenging Savin, et al. 1990). The metabolic selectivity towards bu- tyrate is further increased with the presence of acetate in the fermentations with 26.3 g/L acetic acid exacerbates the media as the acetate pre-adapted culture produced the difference between carbon sources and leads to a Relative Acetic Acid (g/L) production/consumption during xylose fermentation Jaros et al. SpringerPlus 2013, 2:47 Page 8 of 8 http://www.springerplus.com/content/2/1/47 significant increase in butyric acid yield or xylose con- acetate tolerance. As selective adaption is a simpler tech- suming batches (data not shown). Given 26.3 g/L acetic nique to perform than genetic modification, the work here acid inhibition, triplicate non-adapted batches consum- presents the potential for industrially producing all-natural ing 60 g/L xylose generated an average of 30.45 g/L butyric acid for consumer use. butyric acid (+/− 2.80) with duplicate batches of Competing interests challenged glucose consumers producing only 25.20 g/L The authors declare that they have no competing interests. butyric acid (+/− 1.44). Authors’ contributions The overall higher specific growth rates of glucose AMJ performed the strain adaptation, the subsequent fermentations and the batches compared to the xylose batches is another result corresponding analysis. AMJ contributed to the preparation of the of the higher energetic value of glucose metabolism manuscript. UR planned the research strategy of the study and was involved in the interpretation of the data. UR also contributed to the preparation of (Table 1). Due to this, the specific growth rates of the the manuscript. KAB was involved in analysing and interpretation of data glucose batches are all two-to-three times faster than the and preparation of the manuscript. All authors read and approved the final corresponding xylose batches. As would be expected, manuscript. acetate inhibition slows the specific growth rates in glu- Acknowledgments cose batches but surprisingly, the non-adapted acetate The authors gratefully acknowledge the support by the Swedish Energy inhibited xylose batch had a faster specific growth rate Agency, Swedish Governmental Agency for Innovation Systems (VINNOVA), the United States Defense Logistics Agency, and Bio4Energy, a strategic (0.121 1/h) than the control 0.093 1/h (Table 1). This research environment appointed by the Swedish government. can be explained by the long 102 hours of lag-phase that the non-adapted xylose batch had to adapt to the high Received: 11 July 2012 Accepted: 17 December 2012 Published: 11 February 2013 level of acetate. The overall butyric acid productivity of the non-adapted References acetate inhibited xylose batch was only 0.12 g/L/h despite Canganella F, Kuk S-U, Morgan H, Wiegel J (2002) Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate the faster specific growth rate. For industrial practices, the supplementation. Microbiol Res 157:149–156 102 hour lag-phase of the non-adapted xylose batch to Helmerius J, Walter JV, Rova U, Berglund KA, Hodge DB (2010) Impact of start consumption is far too long a period of inactivity. hemicellulose pre-extraction for bioconversion on birch Kraft pulp properties. Bioresour Technol 101:5996–6005 The week of non-growth as the non-selected culture Herrero AA, Gomez RF (1980) Development of Ethanol Tolerance in Clostridium undergoes lag-phase would tie up fermentation capacity thermocellum: Effect of Growth Temperature. Appl Environ Microbiol 3:571–577 and potentially allow for contamination of the batch with Jaros AM, Rova U, Berglund KA (2012) The Effect of Acetate on the Fermentation Production of Butyrate. Cellulose Chemistry and Technol 5–6:341–347 other acetate tolerant microbes. The acetate adapted C. Lin Y-L, Blaschek HP (1983) Butanol Production by a Butanol-Tolerant Strain of tyrobutyricum culture required only a 25 hour lag-phase Clostridium acetobutylicum in Extruded Corn Broth. Appl Environ Microbiol until xylose consumption began, greatly reducing the time 3:966–973 Liu X, Zhu Y, Yang S-T (2006) Butyric acid and hydrogen production by involved in complete batch fermentation. Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme Microb The final yield of the selected acetate- challenged culture Technol 38:521–528 is 0.48 g/g (butyric acid/xylose), 0.05 g/g higher than con- Madigan MT, Martinko JM, Dunlap PV, Clark DP (2009) Metabolic Diversity: Catabolism of Organic Compounds. In: Brock TD (ed) Biology of trol (0.43 g/g) (Table 2). This indicates the power of a sim- Microorganisms, 12th, Editionth edn. Pearson Education, San Francisco, CA ple selection method to adapt a culture which increases Michel-Savin D, Marchal R, Vandecasteele JP (1990) Control of the selectivity of yield without the use of genetic modification. As one of butyric acid production and improvement of fermentation performance with Clostridium tyrobutyricum. Appl Microbiol Biotechnol 32:387–392 the markets for bacterially fermented butyrate is as an all- Rose IA (1955) Acetate Kinase of Bacteria (Acetokinase). Methods Enzymol 1:591–595 natural food enhancer, a production process not utilizing Shuler ML, Kargi F (2002) How Cells Grow. In: Amundson NR (ed) Bioprocess genetically modified organisms might be a requirement. Engineering Basic Concepts, 2nd edn. Prentice Hall PTR, Upper Saddle River, New Jersey Teleman A, Tenkanen M, Jacobs A, Dahlman O (2002) Characterization of Conclusion O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech. A simple selective adaptation for acetate tolerance ge- Carbohydr Res 4:373–377 Zhang C, Yang H, Yang F, Ma Y (2009) Current Progress on Butyric Acid nerated a C. tyrobutyricum culture capable of reducing the Production by Fermentation. Curr Microbiol 59:656–663 acetate induced lag-phase by 75% for a xylose consuming Zhu Y, Yang S-T (2003) Adaptation of Clostridium tyrobutyricum for Enhanced fermentation and completely negated lag-phase in a glu- Tolerance to Butyric Acid in a Fibrous-Bed Bioreactor. Biotechnol Prog 19:365–372 Zhu Y, Yang S-T (2004) Effect of pH on metabolic pathway shift in fermentation cose batch. Specific growth rates for acetate inhibited of xylose by Clostridium tyrobutyricum. J Biotechnol 110:143–157 (26.3 g/L) batches of adapted cultures were reduced com- Zhu Y, Liu X, Yang S-T (2005) Construction and Characterization of pta Gene- pared to non-inhibited control batches but despite this, the Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid Fermentation. Biotechnol Bioeng 2:154–166 adapted cultures demonstrated greater overall butyric acid production than controls for either carbon source. Enzym- doi:10.1186/2193-1801-2-47 atic data collected on acetate kinase demonstrated reduced Cite this article as: Jaros et al.: Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate. activity in cultures fermenting xylose in the presence of SpringerPlus 2013 2:47. acetate whether or not the culture had been selected for http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png SpringerPlus Springer Journals

Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate

SpringerPlus , Volume 2 (1) – Feb 11, 2013

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Abstract

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium capable of utilizing xylose for the fermentation production of butyrate. Hot water extraction of hardwood lingocellulose is an efficient method of producing xylose where autohydrolysis of xylan is catalysed by acetate originating from acetyl groups present in hemicellulose. The presence of acetic acid in the hydrolysate might have a severe impact on the subsequent fermentations. In this study the fermentation kinetics of C. tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate equivalents were studied. Analysis of xylose batch fermentations found that even in the presence of high levels of acetate, acetate adapted strains had similar fermentation kinetics as the parental strain cultivated without acetate. The parental strain exposed to acetate at inhibitory conditions demonstrated a pronounced lag phase (over 100 hours) in growth and butyrate production as compared to the adapted strain (25 hour lag) or non-inhibited controls (0 lag). Additional insight into the metabolic pathway of xylose consumption was gained by determining the specific activity of the acetate kinase (AK) enzyme in adapted versus control batches. AK activity was reduced by 63% in the presence of inhibitory levels of acetate, whether or not the culture had been adapted. Keywords: Clostridium tyrobutyricum, Butyrate, Xylose fermentation, Hemicellulose utilization, Acetate inhibition Introduction microbial growth (Helmerius, et al. 2010). When used in Butyric acid is approved by the Food and Drug Adminis- fermentation media, the inhibitory acetate generates a tration (US) as a flavor enhancer and several flavor esters long lag period before log phase growth and butyric acid used in the food industry are derived from butyric acid. production (Jaros, et al. 2012). Previous work has shown There is a well established market for all-natural foods, that addition of 17.6 g/L and 26.3 g/L acetate in the media where the components are not synthetically derived from generates a lag phase of 45 and 118 hours respectively petro-chemicals as well as a strong consumer bias against while un-inhibited controls begin fermentation and subse- using genetically modified organisms (GMOs) in food quently production almost immediately upon inoculation production. Due to this, butyric acid fermented from bio- (Jaros, et al. 2012). mass by wild type anaerobic bacteria can be developed as Multiple Clostridial strains have been classically selected a saleable commodity. for increased tolerance to both butanol and ethanol which Un-utilized hemicellulose streams from the pulp and successfully lead to higher solvent yields and higher over- paper industry can potentially, after hydrolysis, provide all productivity (Lin and Blaschek 1983; Herrero and a low-cost source of xylose feedstock for organic acid Gomez 1980). Due to the toxicity of these compounds, fermentation. Hardwood xylan is extensively acetylated, each step of the selection requires a short unchallenged i.e. up to seven acetyl groups per ten xylose units which incubation period, an exigency removed when challenging facilitate xylose release by autohydrolysis (Teleman, et al. the organism with acetate. 2002). The resulting hemicellulose hydrolysate contains For organic acid production, non-solventogenic Clostridia levels of acetate of up to 40 g/L acetic acid, inhibitory to such a C. tyrobutyricum are used in fermentation processes where none of the typical toxic by-products such as butanol and ethanol are produced. C. tyrobutyricum cultures have * Correspondence: Ulrika.Rova@ltu.se Luleå University of Technology, Luleå SE-971 87, Sweden been selectively adapted to tolerate the presence of Michigan State University, East Lansing, MI 48824, USA © 2013 Jaros et al.; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jaros et al. SpringerPlus 2013, 2:47 Page 2 of 8 http://www.springerplus.com/content/2/1/47 inhibitory organic acids in order to increase acid product fermenting wild type cultures (Jaros, et al. 2012). This sim- yields (Zhu and Yang 2003). Despite their success, these ple means of directing carbon flux towards butyric acid selections have been performed on immobilized production is an added benefit of working with high acetate C. tyrobutyricum cultures in fibrous-bed bioreactors media and is especially important in light of evidence that requiring a 3 day cell growth period followed by a 36 to such levels of acetate are present in potential xylose feed- 48 hour cell immobilization period in order for a conti- stock streams (Helmerius, et al. 2010; Jaros, et al. 2012). nuous feed fermentation to begin (Zhu and Yang 2003). C. tyrobutyricum batch fermentations under high acetate Such a process allows for the eventual in-line adaptation challenged conditions perform better with xylose as a car- of a C. tyrobutyricum culture to inhibitory acid products bon source than glucose. Fermentations with 26.3 g/L ini- while simple adaptation techniques produce a tole- tial acetate generated 32.6 g/L butyric acid on xylose, while rant culture ready to inoculate immediately into the comparable batch with glucose feed produced 22.3 g/L batch fermentation. (Jaros, et al. 2012). Similar results were received with all Through our work we have detected that C. tyrobutyricum initial acetate concentrations (0, 4.4, 8.8, 17.6 g/L). How- demonstrates diauxic growth, the phenomena of a meta- ever batch fermentations utilizing high acetate (26.3 g/L bolic shift occurring in the middle of the growth cycle initial acetate) xylose synthetic media resulted in an when the two carbon sources glucose and xylose are pre- extended lag phase of 118 hours, lowering productivity sent (data not shown). The presence of a more utilizable (Jaros, et al. 2012). The extended lag phase generated by carbon source, in this case, glucose, prevents activation acetate is economically detrimental for batch fermentation of the metabolic machinery required for the cells to of butyrate as it leads to a long period of reactor inactivity consume the secondary substrate, xylose. Fortunately, and potential exposure to microbial contamination. On the C. tyrobutyricum readily consumes xylose if the culture other hand, after lag phase the 26.3 g/L initial acetate has been pre-conditioned to xylose metabolism and no challenged batch obtained a similar biomass concentration other sugar sources are available. as the lower acetate and control batches and surpassed Anaerobic, butyrate producing bacteria such as Clostridia them in final butyrate yield (Jaros, et al. 2012). The focus metabolize glucose to pyruvate through the Embden- of this work is to adapt a strain of C. tyrobutyricum to in- Meyerhof-Parnas (EMP) pathway and concomitantly creased acetate tolerance, thus decreasing the extended lag generate acetate, butyrate, H and CO as major meta- phase while maintaining the acetate re-utilization meta- 2 2 bolic end-products (Zhang, et al. 2009). Xylose is speci- bolic mechanism to deliver increased yields of butyric acid. fically catabolised in the Hexose Monophosphate Pathway As hardwood derived hemicellulose hydrolysate feedstock to pyruvate which is enzymatically co-oxidized with cellu- gives rise to high levels of both xylose and acetate, a xylose lar coenzyme-A to acetyl coenzyme A (Zhu and Yang consuming strain capable of overcoming the acetate in- 2004; Madigan, et al. 2009). Acetyl-CoA is the branch- duced lag and yet re-utilizing acetate to generate even point node of the acetate and butyrate end-product path- more butyric acid would be of commercial value. ways where the enzymes phosphotransacetylase (PTA) and acetate kinase (AK) are responsible for the metabo- Methods lism of acetyl-CoA to acetate if the branch-point does not Microorganism and adaptation follow the butyrate pathway (Zhu and Yang 2004). In A lyophilized stock culture of C. tyrobutyricum (ATCC attempts to force the carbon flux from the acetate to bu- 25755) was re-hydrated under sterile anaerobic conditions tyrate metabolic branch in C. tyrobutyricum, mutants have in Reinforced Clostridial Media (RCM; Difco). Once the been developed with inactivation’sin the pta and ack culture entered log phase, when the optical density (OD) genes coding for PTA and AK respectively (Zhu, et al. at 600 nm was approximately 2.0, transfers were made 2005; Liu, et al. 2006). Fermentations with the mutants to glycerol stock vials (CRYOBANK ) and the culture yielded more butyric acid compared to wild type was maintained at −70°C. C. tyrobutyricum was classically C. tyrobutyricum, but both mutant strains demonstrated adapted to 26.3 g/L inhibitory acetate equivalents by serially significantly slower growth kinetics than wild type and in passaging log phase cultures into serum bottles with RCM both cases resulted in higher final acetic acid concentrations containing subsequently higher concentrations of sodium with increased acid tolerance (Zhu, et al. 2005; Liu, et al. acetate (starting at 0 g/L then, 6 g/L, 12 g/L, 24 g/L and 2006). These results exhibit a common issue of genetic 36 g/L sequentially) at each passage. As the molar mass of engineering in that GMO’s are typically less robust than sodium acetate is 82.03 g/mol, these concentrations corres- wild type (slower growth) and the complexity of most pond with 0 g/L, 4.4 g/L, 8.8 g/L, 17.6 g/L, and 26.3 g/L metabolic pathways allows for the re-routing of acetic acid equivalents respectively. inactivated processes due to homeostasis. The presence of The adaptation was performed on two sets of C. 17.6 g/L to 26.3 g/L initial acetate in the media has the tyrobutyricum cultures, each culture solely conditioned similar effect of lowering acetate production in xylose to consuming either xylose or glucose so that the actual Jaros et al. SpringerPlus 2013, 2:47 Page 3 of 8 http://www.springerplus.com/content/2/1/47 batch fermentations could be performed without a lag fermentation. Sodium acetate (0 – 36 g/L) was added to phase due to an altered sugar source. The glucose con- the initial media prior inoculation for studies assessing ditioned culture was maintained with RCM from Difco acetate inhibition. Fermentations without acetate are re- with the appropriate additions of acetate equivalents in ferred to as controls. Samples (10 mL) were withdrawn at the form of sodium acetate. The xylose conditioned cul- regular intervals for analytical measurements. Data ture bottles also received the appropriate amount of ace- presented in the tables and figures of this study are the tate equivalent from a media consisting of: 10 g peptone results of single batch fermentations while an analysis in- (Fisher), 10 g beef extract (Teknova), 3 g yeast extract volving duplicate and triplicate fermentations is given in (Bacto), 5 g sodium chloride (J.T. Baker), 0.5g L-cysteine the discussion where stated. (Sigma-Aldrich), 3g sodium acetate anhydrous (J.T. Baker), 0.5 g agar (Bacto) and 900 mL distilled water. For the Analytical methods xylose feed, 5 g of xylose (Acros) in 10 mL distilled water, Organic acids and residual sugar were analyzed by HPLC separately autoclaved at 121°C for 20 min was added to the (LC-20AT dual pump and 10A RI detector, Shimadzu) culture media. Prior to autoclaving all serum bottles were equipped with an ion exchange column (Aminex HPX- sparged with nitrogen to maintain an anaerobic atmos- 87H, 9 um, 7.8 mm x 300 mm, Bio-Rad) and a cation-H phere. Each serum bottle contained a total volume of 100 guard column (Micro-guard, 30 mm × 4.6 mm) using 50 mL RCM (initial pH 6.5) with 5 mL from the previous mM sulfuric acid as a mobile phase. The flow rate of the stage used to inoculate the next higher acetate stage. Du- mobile phase was maintained at 1 mL/min during analysis ring adaptation, serum bottles were incubated at 36°C in with 20 μL of sample injected into the system with an an incubator-shaker (New Brunswick Scientific Innova 40) auto-injector (SIL-20AHT, Shimadzu) with the column with shaking at 80 rpm. and guard maintained at 65°C in a column oven (CT0- The cultures required 24 hours to adapt and reach log 20A, Shimadzu). Prior to analyses, samples were centri- phase growth before passaging to the next level of selec- fuged at 10 000 rpm for 5 min in a micro-centrifuge tion with the exception of the last transfer of the 17.6 g/L (Microfuge 18, Beckman Coulter). Data for each sample acetate adapted cultures to the final 26.3 g/L. Glucose was acquired with Shimadzu EZ Start 7.4 SP1 chromatog- conditioned cultures required 48 hours to reach log phase raphy software using standards for glucose, xylose, butyr- when challenged with 26.3 g/L acetic acid and xylose ate, acetate and lactate. conditioned required 96 hours of incubation to reach log phase. C. tyrobutyricum inoculum for each batch fermenta- Dry cellular weight determination tion were pre-conditioned to the correct sugar substrate Cell growth was monitored during fermentation by meas- in the inoculation media prior the batch fermentation by uring the optical density at 600 nm. The biomass from 40 anaerobically inoculating 50 mL Screw Cap Corning mL cell suspension, removed in triplicate, was dried in an tubes containing 35 mL sterile glucose or xylose based 80°C drier for 48 hours and the dry cell weight (DCW, g/L) RCM with 5 mL of the stock culture. The inoculated tubes determined. The optical densities were then converted to were cultivated under anaerobic conditions at 36°C, 80 dry cell weight using the following equation: DCW = 0.38 rpm, until log phase, approximately when OD had (OD ). This optical density to dry cellular weight conver- reached a value of 2. 600 sion formula was determined for the specific organism and media used in this study. Fermentations One liter batch fermentations were conducted in New Brunswick Bioflo 310 2.5 L working volume reactors under anaerobic conditions at 36°C. For each batch, 950 Specific Growth Rate (μ ) net mL media of the following composition was used; 6 g/L DCW was used to determine the specific growth rate as yeast extract, 5 ppm FeSO 7H O, and 200 mL xylose described by Shuler et al. (Shuler and Kargi 2002). The 4 2 or glucose at 300 g/L sterilized separately. Anaerobiosis DCW data points from the logarithmic growth phase was reached by sparging the vessel with nitrogen prior were plotted on a semi-log graph to locate the period to inoculation. The batches were inoculated with 50 mL during that phase in which the culture experienced the log phase C. tyrobutyricum cultures. The nitrogen spar- fastest growth. These points were then used in the ging was maintained until logarithmic growth in the ves- following equation: μ (1/h) = (ln(DCW /DCW ))/ net x 0 sel was observed. (Time -Time ), where DCW was measured in g/L and x 0 Agitation was kept at 250 rpm and in order to maintain time in hours. DCW is the last point during the fasted the C. tyrobutyricum cultures in acidogenic production, logarithmic growth period and DCW is the first point. pH 6.0 was sustained with 5 M NaOH throughout the Time and Time are described similarly. x 0 Jaros et al. SpringerPlus 2013, 2:47 Page 4 of 8 http://www.springerplus.com/content/2/1/47 Acetate kinase assay consumption almost immediately with butyric acid pro- Bacterial cells from xylose conditioned batches at log phase duction beginning 15 hours later (Figure 1a and 1b). growth were chilled on ice and centrifuged at room The same culture inoculated into xylose-minimal media temperature at 5,000 rpm for 5 min and washed in 25 mM containing 26.3 g/L acetate equivalents required over Tris–HCl, pH 7.4 in order to remove acetate from the 100 hours to acclimate to the acetate despite both medium. After a second centrifugation the cell pellet was fermentations operating under the same conditions. The resuspended in 25 mM Tris–HCl, pH 7.4 and sonicated extended period of minimal metabolism and productivity three intervals at 30 khz for 60 seconds, while on ice, to is due to the acetate causing a delay in log phase cellular lyse the cell wall. The supernatant was used for acetate ac- growth (Figure 1c). Once the C. tyrobutyricum culture tivity studies using a method (Rose 1955) where the con- had adapted to the 26.3 g/L acetate media the culture version of acetate to acyl phosphates by acetate kinase is performed like the control, resulting in complete xylose coupled to the formation of a ferric-hydroxamate complex utilisation and production of over 25 g/L of butyric acid detectable by UV–visat540 nm.In summary,the enzyme and similar levels of cell mass. activity was measured at 29°C using UV/VIS spectroscopy The acetate adapted culture maintained tolerance to where the absorbance of a 4 mL reaction mixture at the 26.3 g/L acetate in the media and after a 22 hour lag 540 nm and the ferric-hydroxamate complex molar extinc- in xylose consumption following inoculation, subse- -1 -1 tion coefficient of 0.169 mM cm was used to calculate quently began producing butyric acid (Figure 1a and 1b). the enzyme activity (Zhu and Yang 2003; Zhu, et al. 2005). The acetate tolerant culture running under acetate in- Acetate kinase activity was standardized to the total pro- hibition conditions performed similar to the control fer- tein content of each sample, determined separately by mentation in that the xylose was fully utilized in 175 Bradford (Bio-rad protein assay) using bovine serum albu- hours from inoculation and produced 28 g/L butyric min. One unit of acetate kinase is defined as the amount of acid compared to the controls production of 25.8 g/L enzyme producing 1 μmol of hydroxamic acid per minute butyric acid. Despite the increased product yield, the net at 29°C and the specific activity calculated as units of specific growth rate (μ ) of the acetate tolerant culture net activity/mg cellular protein.The resultsreportedhere was reduced by 28.7% compared to the control. The are averages of enzyme assays run in triplicate. specific growth rate of the control fermentation was 0.093 1/h while the acetate selected culture showed a Results log phase growth of 0.067 1/h (Table 1). This observa- Fermentation kinetics tion is not surprising as a similar yield increase The non-adapted (control) C. tyrobutyricum culture corresponding with a growth rate reduction was seen in inoculated into xylose-minimal media begins sugar genetically modified C. tyrobutyricum where the pta No external acetate with non-adapted culture 26.3 g/L external acetate with adapted culture 26.3 g/L external acetate with non- adapted culture 0100 200 300 Time (h) bc 35 3.5 30 3.0 25 2.5 20 2.0 15 1.5 10 1.0 5 0.5 0 0.0 0100 200 300 0 100 200 300 Time (h) Time (h) Figure 1 Impact of acetate on xylose consumption, butyric acid production and biomass generation. Butyric Acid equivalents Xylose (g/L) (g/L) DCW (g/L) Jaros et al. SpringerPlus 2013, 2:47 Page 5 of 8 http://www.springerplus.com/content/2/1/47 Table 1 Fermentation kinetics of C. tyrobutyricum cultures run in batch with or without selection for acetate tolerance and with or without acetate inhibition Sugar Acetate C. Lag Complete Sugar Butyrate Final concentration Specific Growth Overall 2 3 4 5 7 8 tyrobutyricum time utilization of cons Yield Rate (μ ) produc. net carbon (g/L) (h) (h) (g/L/h) (mol/mol) (g/L) (1/h) (g/L/h) Butyrate Acetate Bio- mass Glc 0 non-adapted 0 77 1.07 0.85 25.61 8.38 3.40 0.306 0.28 Glc 26.3 non-adapted 94 171 1.09 0.89 26.22 27.85 3.59 0.274 0.15 Glc 26.3 adapted 0 75 1.21 0.87 25.86 32.03 2.77 0.206 0.32 Xyl 0 non-adapted 0 166 0.56 0.74 25.80 4.24 2.72 0.093 0.16 Xyl 26.3 non-adapted 102 167 1.22 0.79 29.00 27.76 3.04 0.121 0.12 Xyl 26.3 adapted 25 174 0.60 0.81 28.92 24.46 2.30 0.067 0.17 Glucose and xylose respectively. Whether or not the inoculum had been selectively adapted to 26.3 g/L. Calculated as time until sugar consumption started. Calculated for the linear sugar consumption phase. Yield was calculated as mol butyrate per mol glucose or xylose consumed during fermentation. Calculated as DCW g/L. 7 -1 As determined by the formula μnet (h ) = (ln(DCW /DCW ))/(Time -Time ). x 0 x 0 Overall productivity calculated from the start of the fermentation until the sugar source were completed. gene had been deleted (Zhu, et al. 2005; Liu, et al. non-adapted-inhibited and adapted-inhibited) generated 2006). very similar levels of butyric acid between batches (25.61, The effectiveness of selective adaptation to generate an 26.22 and 25.86 g/L respectively) (Table 1). Analogous to acetate tolerant C. tyrobutyricum culture is even more the xylose batches, the acetate inhibited non-adapted cul- evident in glucose consuming fermentations. The adapted ture experienced approximately 94 hours of lag phase be- inoculum under 26.3 g/L acetate conditions experienced fore beginning to consume glucose, produce butyric acid no lag in growth and tracked almost exactly with the unin- or generate DCW biomass (Figure 2a-c, Table 1). Acetate hibited control in terms of glucose consumption and adaptation allows the culture to overcome inhibition butyric acid production (Figure 2a and 2b). Unlike the caused by 26.3 g/L acetate and the 94 hours of lag phase. xylose batches, the glucose consuming cultures (control, A net production of acetate occurred in the glucose No external acetate 60 with non-adapted culture 26.3 g/L external acetate with 30 adapted culture 20 26.3 g/L external acetate with non- adapted culture 0100 200 300 Time (h) bc 30 4 3.5 2.5 15 2 1.5 0.5 0 0 0100 200 300 0 100 200 300 Time (h) Time (h) Figure 2 Impact of acetate on glucose consumption, butyric acid production and biomass generation. Butyric Acid equivalents Glucose (g/L) (g/L) DCW (g/L) Jaros et al. SpringerPlus 2013, 2:47 Page 6 of 8 http://www.springerplus.com/content/2/1/47 consuming acetate adapted batch demonstrating the shown), compared to 94 hours for the non-adapted higher cellular energy made available from glucose con- strain. In contrast, there was a complete reversion of sumption as compared to that of xylose. The xylose con- the acetate adapted strain during xylose fermentation suming acetate adapted batch activated the Clostridial usinganinoculumfromcryogenic storage. Further acetate re-utilization pathway resulting in an overall con- characterization of strain stability and the molecular sumption of acetate rather than production. This activa- mechanisms resulting in increased tolerance for acetate tion was likely necessitated by the lower amount of energy is needed to identify target enzyme pathways or individ- from xylose metabolism. ual genes important for the desired phenotype. The Similar to the xylose batches, the acetate tolerant culture induced tolerance of C. tyrobutyricum enables one to consuming glucose also exhibited a 32.7% reduction in spe- use adaptation as a tool to identify alteration of the cific growth rate compared with the glucose control culture organism's own enzyme systems that can be targeted for (Table 1). The glucose control batch demonstrated a 0.306 further permanent genetic modification. 1/h specific growth rate and the adapted culture dropped to 0.206 1/h during acetate inhibition (26.3 g/L). The non- Acetate kinase activity adapted culture under acetate inhibition (26.3 g/L) dropped The metabolic selectivity in C. tyrobutyricum is influenced to 0.274 1/h, only a 10.5% reduction compared to the glu- by growth stage, with exponentially growing cultures pro- cose control batch. ducing both butyric and acetic acids, while slower station- The specific growth rates of glucose consuming ary growth rates tend towards butyric acid (Michel-Savin, batches were two to three times higher than those of the et al. 1990). As such, during log phase growth of each xylose consuming C. tyrobutyricum batches (Table 1). batch, culture samples were removed and analyzed for Lowered specific growth rates are a consequence of xy- acetate kinase activity. Acetate kinase (AK) is the last en- lose consumption due to the lowered energetic value of zyme on the metabolic arm converting acetyl-CoA to xylose metabolism over glucose. With less free energy acetate, thus AK activity under particular fermentation from sugar consumption, the xylose consuming batches conditions is related to acetate production (Liu, et al. have less energy to perform cellular maintenance and 2006). Table 3 presents the specific activity in relation growth thus, in general have lower specific growth rates to cellular protein. The presence of inhibitory acetate than glucose consuming batches. (26.3 g/L) in the media reduced the AK activity to 3.15 The xylose consuming acetate-inhibited batches exhibited U/mg in both the adapted and non-adapted cultures as higher final yields of butyric acid than the control culture the control culture exhibited 8.42 U/mg (Table 3). In (Table 2). Both the acetate tolerant and non-adapted both cases of acetate inhibition, whether the culture was cultures yielded 0.48 g/g butyric acid from the initial 60 g/L acetate tolerant or not, the acetate kinase activity was xylose compared to the control cultures 0.43 g/g. Glucose reduced leading to the inhibition of metabolic acetate consuming cultures demonstrated no significant change in production (Figure 3, Table 1). butyric acid yield between the 3 batches (Table 2). The AK specific activity results correlate strongly to The selection pressure during cultivation in 26.3 g/L the production data in Figure 3, where the control cul- acetate medium with xylose or glucose resulted in a ture with the highest AK activity also generated the strain with improved butyrate production while exposed most acetic acid equivalents, 4.24 g/L. The non-adapted to high acetate concentrations during fermentation. batch with 26.3 g/L initial acetic acid equivalents and However, this phenotype was only preserved to some the lowered AK activity generated only an additional extent for the glucose fermenting acetate adapted strain. 2.65 g/L acetic acid by the time the xylose had been When this adapted strain, stored at −70°C, was used dir- completely utilized (Figure 3). The selected batch run ectly to inoculate a 26.3 g/L acetate challenged media, under the same initial acetic acid conditions performed the lag phase was increased to 42 hours (results not Table 3 The impact of the presence of acetate on enzymatic Acetate Kinase activity in C. tyrobutyricum Table 2 The effect of acetate inhibition on butyric acid fermentations yield in batch fermentations of C. tyrobutyricum with an No external 26.3 g/L 26.3 g/L initial 60 g/L glucose or xylose and run until completion acetate with external external Butyric acid yield (g/g) non-adapted acetate with acetate with culture non-adapted adapted Carbon No external 26.3 g/L external 26.3 g/L external culture culture source acetate with acetate with non- acetate with non-adapted adapted culture adapted culture Acetate Kinase 8.42 3.15 3.15 culture activity (Units/mg cellular protein) Glucose 0.43 0.44 0.43 Results reported here are averages of enzymes assays run in triplicate as Xylose 0.43 0.48 0.48 described in the methods. Jaros et al. SpringerPlus 2013, 2:47 Page 7 of 8 http://www.springerplus.com/content/2/1/47 negligible quantities of acetic acid even during the begin- ning log phase stage (Figure 3). Other than AK inhibition, another innate mechanism No external acetate with pushing the carbon flux of the Clostridial metabolism non-adapted culture towards butyrate and away from acetate is the re-uptake 26.3 g/L external acetate of acetate from the media back into the usable acetyl- with acetate adapted culture 0100 200 300 CoA pool by the CoA transferase enzyme (Michel-Savin, -1 26.3 g/L external acetate with non-adapted culture et al. 1990). This re-utilization mechanism of acetate -2 provides no energy benefits to the cell but allows for the -3 control of environmental acetate and utilizes protons in the -4 Time (h) acetate-to-butyrate conversion process (Michel-Savin, et al. Figure 3 Effect of acetate inhibition on relative acetic acid 1990). Acetate re-uptake can be exploited under the fermentation kinetics of C. tyrobutyricum xylose batches. conditions pertaining to a microbial inhibiting level of acetate present in the feed stream since the supposed con- with even higher carbon flux away from the acetate taminant in this case can potentially be used as a carbon branch as acetate re-uptake mechanisms allowed the source (Helmerius, et al. 2010; Jaros, et al. 2012). Some of culture to consume 2.47 g/L of the initial acetate from the re-assimilated acetyl-CoA enters the butyrate pathway the media (Figure 3). and thus this mechanism contributes to carbon efficiency (Canganella, et al. 2002). Acetate re-uptake occurred in Discussion the xylose consuming pre-adapted fermentation, not Acetate tolerant C. tyrobutyricum cultures consuming only is the final butyric concentration (28.92 g/L) higher xylose overcame the acetate induced lag growth phase than the control (25.8 g/L) but the initial acetate con- four times faster than the comparable non-selected centration decreases during the course of the study cultures under the same acetate inhibition conditions (Figure 1b and 3). Unfortunately, CoA transferase is also (26.3 g/L) (Figure 1a-c, Table 1). The selected culture implicated in a redundant pathway leading to acetate gen- also maintained lowered utilization of the acetate meta- eration directly from acetyl-CoA, so information concer- bolic pathway under challenged conditions (Figure 3 and ning this enzymes specific activity may not provide useful Table 3). The acetate producing metabolic pathway yields information concerning the acetate re-uptake mechanism more ATP than the butyrate pathway, so an inhibition of (Liu, et al. 2006). acetate kinase (AK) or phosphotransacetylase (PTA) leads The selective adaptation of acetate tolerant glucose to increased carbon flux towards phosphotransbutyrylase consuming cultures completely eliminated the acetate (PTB) and butyrate kinase (BK) as the butyrate pathway induced lag phase in growth under inhibitory conditions must compensate for the energy loss (Zhu and Yang 2004; (Figure 2a-c, Table 1). The higher energetic value of glu- Michel-Savin, et al. 1990). Rather than lower energy con- cose consumption over that of xylose appears to allow sumption and less biomass generated, the acetate inhibited acetate selected cultures consuming glucose to begin C. tyrobutyricum cultures generated a similar amount of fermentation immediately even under 26.3 g/L acetate biomass as the control by increasing butyrate production inhibition (Figure 2a). This is remarkable given that the to overcome the energy inefficiency (Figure 1c, 2c). Similar non-selected glucose consuming batch still required a 94 to our results, C. tyrobutyricum fermentations with genetic hour lag-phase to overcome acetate inhibition, similar to inactivation of pta also had higher butyric yields and the 102 hours seen in the xylose consuming non- inactivated (or in our case, inhibited) acetate producers still selected culture under the same conditions (Table 1). developed similar levels of biomass as controls (Figure 1c, The selective adaptation of C. tyrobutyricum for acetate tolerance is more effective for glucose consuming 2c) (Zhu, et al. 2005). Both acetate kinase and phosphotransacetylase are more cultures than xylose consumers. sensitive to product inhibition by butyrate than the The energetic differences between xylose and glucose consumption appear to also affect the final butyric acid enzymes responsible for the butyrate pathway, butyrate kinase and phosphotransbutyrylase (Zhu and Yang 2003). yields for 26.3 g/L acetate inhibited batches (data not This natural inhibition is beneficial from an industrial shown). Duplicate fermentations of 60 g/L xylose produced an average of 27.16 g/L butyric acid with a standpoint as shortly after the culture enters the exponen- tial growth phase C. tyrobutyricum stops co-producing standard deviation of (+/− 1.93) while duplicate fer- both acid products and singularly forms butyrate (Michel- mentations of 60 g/L dextrose average 24.34 g/L butyric acid (+/− 0.99), a non-significant difference. Challenging Savin, et al. 1990). The metabolic selectivity towards bu- tyrate is further increased with the presence of acetate in the fermentations with 26.3 g/L acetic acid exacerbates the media as the acetate pre-adapted culture produced the difference between carbon sources and leads to a Relative Acetic Acid (g/L) production/consumption during xylose fermentation Jaros et al. SpringerPlus 2013, 2:47 Page 8 of 8 http://www.springerplus.com/content/2/1/47 significant increase in butyric acid yield or xylose con- acetate tolerance. As selective adaption is a simpler tech- suming batches (data not shown). Given 26.3 g/L acetic nique to perform than genetic modification, the work here acid inhibition, triplicate non-adapted batches consum- presents the potential for industrially producing all-natural ing 60 g/L xylose generated an average of 30.45 g/L butyric acid for consumer use. butyric acid (+/− 2.80) with duplicate batches of Competing interests challenged glucose consumers producing only 25.20 g/L The authors declare that they have no competing interests. butyric acid (+/− 1.44). Authors’ contributions The overall higher specific growth rates of glucose AMJ performed the strain adaptation, the subsequent fermentations and the batches compared to the xylose batches is another result corresponding analysis. AMJ contributed to the preparation of the of the higher energetic value of glucose metabolism manuscript. UR planned the research strategy of the study and was involved in the interpretation of the data. UR also contributed to the preparation of (Table 1). Due to this, the specific growth rates of the the manuscript. KAB was involved in analysing and interpretation of data glucose batches are all two-to-three times faster than the and preparation of the manuscript. All authors read and approved the final corresponding xylose batches. As would be expected, manuscript. acetate inhibition slows the specific growth rates in glu- Acknowledgments cose batches but surprisingly, the non-adapted acetate The authors gratefully acknowledge the support by the Swedish Energy inhibited xylose batch had a faster specific growth rate Agency, Swedish Governmental Agency for Innovation Systems (VINNOVA), the United States Defense Logistics Agency, and Bio4Energy, a strategic (0.121 1/h) than the control 0.093 1/h (Table 1). This research environment appointed by the Swedish government. can be explained by the long 102 hours of lag-phase that the non-adapted xylose batch had to adapt to the high Received: 11 July 2012 Accepted: 17 December 2012 Published: 11 February 2013 level of acetate. The overall butyric acid productivity of the non-adapted References acetate inhibited xylose batch was only 0.12 g/L/h despite Canganella F, Kuk S-U, Morgan H, Wiegel J (2002) Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate the faster specific growth rate. For industrial practices, the supplementation. Microbiol Res 157:149–156 102 hour lag-phase of the non-adapted xylose batch to Helmerius J, Walter JV, Rova U, Berglund KA, Hodge DB (2010) Impact of start consumption is far too long a period of inactivity. hemicellulose pre-extraction for bioconversion on birch Kraft pulp properties. Bioresour Technol 101:5996–6005 The week of non-growth as the non-selected culture Herrero AA, Gomez RF (1980) Development of Ethanol Tolerance in Clostridium undergoes lag-phase would tie up fermentation capacity thermocellum: Effect of Growth Temperature. Appl Environ Microbiol 3:571–577 and potentially allow for contamination of the batch with Jaros AM, Rova U, Berglund KA (2012) The Effect of Acetate on the Fermentation Production of Butyrate. Cellulose Chemistry and Technol 5–6:341–347 other acetate tolerant microbes. The acetate adapted C. Lin Y-L, Blaschek HP (1983) Butanol Production by a Butanol-Tolerant Strain of tyrobutyricum culture required only a 25 hour lag-phase Clostridium acetobutylicum in Extruded Corn Broth. Appl Environ Microbiol until xylose consumption began, greatly reducing the time 3:966–973 Liu X, Zhu Y, Yang S-T (2006) Butyric acid and hydrogen production by involved in complete batch fermentation. Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme Microb The final yield of the selected acetate- challenged culture Technol 38:521–528 is 0.48 g/g (butyric acid/xylose), 0.05 g/g higher than con- Madigan MT, Martinko JM, Dunlap PV, Clark DP (2009) Metabolic Diversity: Catabolism of Organic Compounds. In: Brock TD (ed) Biology of trol (0.43 g/g) (Table 2). This indicates the power of a sim- Microorganisms, 12th, Editionth edn. Pearson Education, San Francisco, CA ple selection method to adapt a culture which increases Michel-Savin D, Marchal R, Vandecasteele JP (1990) Control of the selectivity of yield without the use of genetic modification. As one of butyric acid production and improvement of fermentation performance with Clostridium tyrobutyricum. Appl Microbiol Biotechnol 32:387–392 the markets for bacterially fermented butyrate is as an all- Rose IA (1955) Acetate Kinase of Bacteria (Acetokinase). Methods Enzymol 1:591–595 natural food enhancer, a production process not utilizing Shuler ML, Kargi F (2002) How Cells Grow. In: Amundson NR (ed) Bioprocess genetically modified organisms might be a requirement. Engineering Basic Concepts, 2nd edn. Prentice Hall PTR, Upper Saddle River, New Jersey Teleman A, Tenkanen M, Jacobs A, Dahlman O (2002) Characterization of Conclusion O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech. A simple selective adaptation for acetate tolerance ge- Carbohydr Res 4:373–377 Zhang C, Yang H, Yang F, Ma Y (2009) Current Progress on Butyric Acid nerated a C. tyrobutyricum culture capable of reducing the Production by Fermentation. Curr Microbiol 59:656–663 acetate induced lag-phase by 75% for a xylose consuming Zhu Y, Yang S-T (2003) Adaptation of Clostridium tyrobutyricum for Enhanced fermentation and completely negated lag-phase in a glu- Tolerance to Butyric Acid in a Fibrous-Bed Bioreactor. Biotechnol Prog 19:365–372 Zhu Y, Yang S-T (2004) Effect of pH on metabolic pathway shift in fermentation cose batch. Specific growth rates for acetate inhibited of xylose by Clostridium tyrobutyricum. J Biotechnol 110:143–157 (26.3 g/L) batches of adapted cultures were reduced com- Zhu Y, Liu X, Yang S-T (2005) Construction and Characterization of pta Gene- pared to non-inhibited control batches but despite this, the Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid Fermentation. Biotechnol Bioeng 2:154–166 adapted cultures demonstrated greater overall butyric acid production than controls for either carbon source. Enzym- doi:10.1186/2193-1801-2-47 atic data collected on acetate kinase demonstrated reduced Cite this article as: Jaros et al.: Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate. activity in cultures fermenting xylose in the presence of SpringerPlus 2013 2:47. acetate whether or not the culture had been selected for

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Published: Feb 11, 2013

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