lactic acid production by lactobacillus pentosus from wood ... - Paptac

LACTIC ACID PRODUCTION BY LACTOBACILLUS PENTOSUS FROM WOOD EXTRACT HYDROLYSATES ABSTRACT

JOHN P. BUYONDO, SHIJIE LIU* Hot-water extracted hemicelluloses from sugar maple wood chips were hydrolyzed by dilute acid at an elevated temperature and concentrated using a nano-filtration membrane process to obtain a fermentable sugar stream containing arabinose, galactose, glucose, mannose, rhamnose, and xylose. Lactobacillus pentosus was directly adapted to using the concentrated wood extract hydrolysate to produce lactic acid. The effect of initial sugar loading was investigated by diluting the concentrated wood hydrolysate to obtain four sugar concentrations: 54.08 g/L, 61.47 g/L, 94.10 g/L, and 129.50 g/L; the media are labelled as med1, med2, med3, and med4 respectively. After 55 hours of fermentation in a 1-L bioreactor at 37°C and pH 6.0, medium med1 had the highest lactic acid yield (Yp/s) of 0.83 g-lactic acid/g-sugar, representing approximately 97.3% of theoretical yield. Acetic acid was produced after glucose was depleted as the main by-product at up to 49% of the obtained lactic acid concentration. Adaptation of an L. pentosus strain to concentrated wood-extract hydrolysate led to a 10-h reduction in fermentation time and a 15.5% increase in lactic acid production. L. pentosus simultaneously utilized both six-carbon and fivecarbon sugars; arabinose, galactose, glucose, and rhamnose were preferably utilized, whereas mannose and xylose were slowly utilized. Acetic acid was also produced after glucose had been completely consumed.

INTRODUCTION

Lactic acid is an industrially important product with a large global market. Worldwide production in 2005 (exclusive of polymers) was 100,000 tons, with a 15% annual growth rate [1]. Food-related applications are the major use of lactic acid in the United States, accounting for approximately 85% of the commercially produced product [2]. Lactic acid is used as a buffering agent, an acidic flavoring agent, an acidulant, and a bacterial inhibitor in many processed foods. Lactic acid and salts are preferred to other acids used in the food industry because they do not dominate other flavours and also act as preservatives [3,4]. Other applications of lactic acid include its use in the chemical industry for de-liming and metal etching and various uses in the pharmaceutical, textile, and cosmetics industries [4]. Currently, the most important application of lactic acid may be its use as a monomer in the production of a biodegradable and biocompatible polymer, polylactic acid (PLA), which is an environmentally friendly alternative to non-biodegradable

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plastics derived from petrochemicals. PLA is a thermo-plastic polyester, is 100% compostable, and has a life cycle which could potentially reduce carbon dioxide levels in the Earth’s atmosphere. Lactic acid can be produced by either microbial fermentation or chemical synthesis. Fermentation offers several advantages over chemical synthesis, including low cost of substrates, low production temperature, low energy consumption, and high product specificity, meaning that fermentation yields optically pure L-(+)- and D-(−)- lactic acid, a desired stereoisomer [1,5,6]. Optically pure lactic acid is required for the production of the highly crystalline PLA which is suitable for commercial uses. These polymers have potential markets in commodity packaging, fabrication of prosthetic devices, and controlled delivery of drugs in humans [1,4]. PLA can also be used to enhance the strength properties of paper sheets made from bleached or unbleached Kraft pulp produced from hot-water extracted and unextracted sugar maple wood chips [7].

In commercial processes, glucose and corn starches have been widely used as substrates for biological lactic acid production. However, this is economically unfavourable because pure sugars have a higher economic value than the lactic acid produced [8]. For biological production of lactic acid to be feasible, cheap raw materials are necessary

JOHN P. BUYONDO

Department of Paper and Bioprocess Engineering, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY, USA , 13210

Journal of Science & Technology for Forest Products and Processes: VOL.1, NO.3, 2011

SHIJIE LIU

Department of Paper and Bioprocess Engineering, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY, USA , 13210 *Contact: [email protected]

SPECIAL BIOREFINERY ISSUE

Fig. 1 - Biochemical steps in the homolactic fermentation of glucose (redrawn based on Bustos et al. [18]).

to meet the large demand of polymer producers and other industrial users at a relatively low cost. Therefore, a cellulosic feedstock (such as wood) should be used rather than refined substrates (glucose, sucrose, etc.) [9,10]. Lignocellulosic biomass offers a favourable alternative as a feedstock for the biological production of lactic acid because it is readily available, has no competing food value, and is less expensive than either cornstarch or sugars. Hemicellulose is the second most abundant carbohydrate source and accounts for 25%–35% of lignocellulosic material. Hemicelluloses are heterogeneous polymers made of pentoses (D-xylose, D-arabinose), hexoses (D-mannose, D-glucose, D-galactose), and sugar acids [11,12]. Hemicelluloses in hardwood primarily contain xylans, while those in softwoods primarily contain glucomannans [13]. Treatment of the hemicellulosic material with dilute acid at an elevated temperature leads to liberation of fermentable sugars [14]. However, in the

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Fig. 2 - Biochemical steps in the heterolactic fermentation of pentoses (redrawn based on Bailey and Ollis [21]).

bioconversion of lignocellulosic sugars to lactic acid, product yield and volumetric productivity are generally low because of the presence of inhibitory compounds such as furfural, 5-hydroxymethyl furfural (HMF), and weak acids, which are generated during the acid hydrolysis step [10,15,16]. Weak acids inhibit cell proliferation by uncoupling and intracellular anion accumulation, and furfural or HMF inactivates cell replication by inhibiting specific enzymes related to glycolysis or inhibitor reduction [16]. To improve the fermentation yields of wood hydrolysates, many methods for detoxifying inhibitory compounds have been investigated, including neutralization, over-liming, evaporation, the use of ion-exchange resins, and activated charcoal adsorption [15]. Bioconversion of hemicellulosic sugars to lactic acid requires a strain which is capable of fermenting sugar mixtures of hexoses and pentoses. Although much research has been focused on genetically

engineering strains that can efficiently utilize both five-carbon and six-carbon sugars [17,18], the recombinant cells involved have a tendency to become genetically unstable on repeated application. The versatility of Lactobacillus pentosus makes it a potential alternative to genetically modified strains. L. pentosus ferments hexoses using the EMP (Embden-Meyerhoff-Parnas) pathway (Fig. 1) and pentoses using the PK (phosphoketolase) pathway (Fig. 2) [19,20]. In this work, wood-extract hydrolysate from sugar maple wood chips was concentrated by a nano-filtration membrane process to generate a mixture of five-and six-carbon hemicellulose sugars. The bacterial strain Lactobacillus pentosus was challenged by different concentrations of wood-extract hydrolysate (WEH) to produce lactic acid in a batch fermentation process. The effect of adaptation of Lactobacillus pentosus cells to concentrated WEH was also examined.


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MATERIALS AND METHODS Production of Hemicellulose Hydrolysate from Sugar Maple Wood Chips

Sugar maple wood chips were extracted by hot water at approximately 160°C for 2 h. The hot-water extraction was carried out in a 1840.5-L digester with a woodto-water ratio of about 1:4. The wood extract was concentrated approximately ten-fold using a nano-filtration membrane. The concentrated wood extract was hydrolyzed at 135°C for 25 min with 1.5% wt sulphuric acid added. After cooling to room temperature, acid-insoluble lignin was separated from the wood-extract hydrolysate by centrifugation (CEPA highspeed centrifuge Z81G, cylinder speed 16,000 rpm, cylinder diameter 125 mm, New Brunswick Scientific, NJ, USA). The wood-extract hydrolysate was neutralized by calcium hydroxide at room temperature, with just enough calcium hydroxide added to neutralize the sulphuric acid. The neutralized hydrolysate (at a pH of approximately 3.5 due to the high concentration of acetic acid) was again freed of solids by centrifugation. The solids-free hydrolysate was then diluted with fresh water and fractionated twice to remove low-molecularweight substances by the nano-filtration membrane process. The membrane used in this study had a molecular weight cutoff of 100 g/mol [22]. Microorganisms and Seed Culture Preparation

The bacterial strain Lactobacillus pentosus (ATCC 8041) used was obtained from the American Type Culture Collection (ATCC). The strain was maintained on MRS agar medium slant and stored at 4°C. The strain was transferred to a fresh medium every three to four weeks. The MRS medium (Difco, Maryland, U.S.A.) contained 10.0 g/L proteose peptone 3, 10.0 g/L beef extract, 5.0 g/L yeast extract, 20.0 g/L dextrose, 1.0 ml/L Tween 80, 2.0 g/L ammonium citrate, 0.1 g/L MgSO4, 0.05 g/L MnSO4, 2.0 g/L K2HPO4, and 5.0 g/L CH3COONa. The

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MRS medium was supplemented by 20 g/L agar to make slant. The seed culture was prepared by picking one or two large colonies from the slant and inoculating them into 50 mL MRS (55 g/L) medium contained in 125-mL screw-capped plastic flasks (NALGENE, Rochester NY, U.S.A.). The effect of adapting the strain to concentrated wood-extract hydrolysate (WEH) before fermentation was also studied. Plated MRS solid medium supplemented with 10% WEH was inoculated with two or three large colonies picked from MRS medium slants and incubated at 37°C for 24 hours. A seed culture of the adapted strain was prepared by picking one or two large colonies from an MRS-WEH medium plate and inoculating them into 50-mL MRS medium supplemented with 20% (v/v) WEH. The seed culture was incubated at 37°C for 15–24 h on a rotary shaker (GYROMAXTM 747R, Amerex Instruments, Lafayette CA, U.S.A.), operating at 150 rpm. The media was sterilized by autoclaving (HIRAYAMA HICLAVETM HV-110, Amerex Instruments, Lafayette CA, U.S.A.) at 121°C for 20 min. Batch Fermentation

Batch fermentation experiments were conducted in a 1.0-L New Brunswick bioreactor (BIOFLO 110; New Brunswick Scientific, New Brunswick NJ, U.S.A.) with an 800-mL working volume. The desired wood hydrolysate concentration was obtained by diluting the concentrated wood hydrolysate with the appropriate volume, V, of distilled water. The fermentation medium contained wood-extract hydrolysate supplemented by 10 g/L yeast extract, 2 g/L K2HPO4, 2 g/L KH2PO4, and 0.5% (v/v) Tween 80. All medium components were purchased from Fisher Scientific, Pittsburgh PA, U.S.A. Wood-extract hydrolysate was sterilized by filtration through a 0.22-µm sterile filter (nitrocellulose membrane, Millipore) which was held on a filter holder with a receiver (1000 mL, NALGENE, Rochester NY, U.S.A.). The fermentation-medium nutrient sup-

plements were dissolved in the desired amount of distilled water in a bioreactor and autoclaved at 121°C for 20 min. After cooling to room temperature, an amount of filtered sterilized concentrated wood-extract hydrolysate was added to the bioreactor and the pH adjusted to 6.0 before inoculation. The bioreactor was inoculated with 40 mL of actively growing 15–24 h-old seed culture and incubated at 37°C. Agitation speed was set to 2.5 Hz, and air flow rate was set to 25 mL/min. The pH was maintained at 6.0 by dosing with a 5 mol/L NaOH solution. Two parallel fermentations were performed, one with native seed culture, and the other with adapted seed culture. Samples (2 mL) were taken at given fermentation times and centrifuged at 4000 rpm for 5 min. The supernatants were stored at -10°C for sugar, lactic acid, and acetic acid analyses. Experimental data were obtained in triplicate, and the values reported represent sample means. Analytical Methods Lactic acid, acetic acid, and inhibitor compound concentrations were determined by 600-MHz 1H-NMR, whereas sugar concentrations were determined by 600-MHz 13 C-NMR (Bruker BioSpin Corporation, Billerica MA, U.S.A.). After centrifugation, samples were prepared by mixing 100 μL sample and 100 μL internal standard (glucosamine, Sigma-Aldrich, St. Louis MO, U.S.A.) with 800 μL D2O. The NMR operating conditions were as follows: probe type: Broadband Observe Probe; temperature: 30°C; 90° pulse: 11 μs; interval between pulses: 10 s; acquisition interval: 2.73 s; sweep width: 10 ppm; center of spectrum: 4.5 ppm; reference: acetone at 2.2 ppm. A calibration curve was generated by plotting peak/area ratios of individual standard solutions and the internal standard. Peak/area ratios (analyte/internal standards) were compared with standard curves to quantify the analytes. The area of each peak was integrated using the MestReNova software, and the integral of glucosamine was set to 100. Lactic acid yield was defined as the amount of acid


SPECIAL BIOREFINERY ISSUE produced divided by the total sugars utilized. RESULTS AND DISCUSSION

ions act as a catalyst to soften the glycosidic bonds and induce their de-polymerization. Glycosidic bond breakage is enhanced when the breakaway oligomers

are attracted away from the solid surface. High temperature and high electrolyte content facilitate the presence of oligomers in the liquor phase [11].

Production of Hemicellulosic Sugars from Sugar Maple Wood Chips

Figures 3 and 4 show the major (xylose) and minor (mannose, glucose, galactose, and rhamnose) monomer and oligomer sugar components produced from sugar maple wood chips during the hot-water extraction process [22]. It can be observed that monomer and oligomer concentrations increased with increasing reaction time. Because of the change in temperature during hot-water extraction, Figs. 3 and 4 show the relative time at 100°C with an activation energy of E/R = 10800 K. Because xylan is the main component of hardwood hemicelluloses, xylo-oligomers and xylose are the main products obtained in hot-water extracts of sugar maple wood chips. In addition, xylan is the easiest component to separate among the three major components of hardwood: cellulose (glucan), hemicellulose (xylan), and lignin. The hemicellulose is extracted from wood chips in the form of xylo-oligomers with various degree of polymerization, and the amount of xylose monomer in the extract is low [23]. Glucomannan hemicellulose is the main source of glucose and mannose. There are acidic compounds in the hot-water extractable portion of the woody biomass. The acetyl groups from hemicellulose, for example, contribute to acetic-acid formation in the extraction liquor or in the wood extracts. The dissolution of acidic components in water causes the liquor pH to drop and effectively generates acid as a catalyst for the extraction [23]. The results obtained here showed that the pH of the hot-water sugar maple wood-chip extracts is approximately 3.5 when samples are cooled and measured at room temperature. The acidic conditions catalyze the extraction and hydrolysis reactions. Therefore, hot-water extraction is referred to as auto-catalytic and sometimes as an auto-hydrolysis process [23]. In an acid hydrolysis reaction, hydrogen

Fig. 3 - Hot-water extraction: minor sugars.

Fig. 4 - Hot-water extraction: xylose and xylo-oligomers.

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Production of Fermentable Hemicellulosic Sugars

Hot-water extract from sugar maple wood chips was hydrolyzed by 1.5% (wt.) concentrated sulphuric acid at elevated temperature (135°C) to obtain fermentable hemicellulosic sugars. After cooling to room temperature, the acidic (pH 0.5) wood-extract hydrolysate was stoichiometrically neutralized (with respect to added sulphuric acid) with calcium hydroxide to approximately pH 3.5. The acid hydrolysis of hot-water extract from sugar maple wood chips led to simultaneous generation of hemicellulosic sugars and fermentation inhibitors such as acetic acid, formic acid, furfural, and hydroxymethyl furfural (HMF). Upon nanofiltration membrane processing, water and other low-molecular-weight compounds such as acetic acid, formic acid, HMF, and furfural were preferentially sent to the permeate stream, while sugar monomers and oligomers along with high-molecularweight compounds were concentrated in the concentrate stream [15]. Figure 5 shows the 2D NMR spectrum for the obtained mixture of hemicellulosic sugars in the sugar maple wood-extract hydrolysate. The beta and alpha sugar components are located between the 5.0–5.3 ppm and 4.4–4.95 ppm chemical shifts respectively. The concentration of the respective sugars was obtained by determining the average of the concentration of the beta and alpha sugar components. After membrane separation, the concentrations of sugars in the wood hydrolysate were 123 g/L xylose, 21.9 g/L mannose, 16.5 g/L glucose, 11.5 g/L galactose, 5.52 g/L arabinose, and 4.13 g/L rhamnose. Here xylose, a five-carbon sugar, is the dominant component of the wood-extract hydrolysate. Arabinose is another five-carbon sugar. Mannose, glucose, and galactose are six-carbon sugars, while rhamnose is a deoxy six-carbon sugar. The ratio of glucose to mannose is close to one, which suggests that their main origin was glucomannan [23]. No formic acid, furfural, or HMF was detected in the concentrated wood-extract hydrolysate, and the concentration of

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Fig. 5 - 2D NMR of standard mixture of monosaccharides.

acetic acid was significantly reduced (Table 1). Fermentation of Wood-Extract Hydrolysate:Preparation of Fermentation Medium

To investigate the possible effect of wood-extract hydrolysate (WEH) concentration on the production of lactic acid, concentrated WEH was diluted with distilled water. Fermentation media were prepared by adding different amounts of distilled water to a known volume of concentrated WEH (mL) to obtain different sugar concentrations (Table

2). The main sugar component of the fermentation media was xylose, contributing about 68% of the total sugar concentration. This agrees with the results presented in the literature by Liu et al. [15] and Amidon et al. [23] that xylose is the main component of sugar maple woodextract hydrolysate. Lactic Acid Fermentation

The seed culture (40 mL) was inoculated into the prepared fermentation medium, and the batch fermentation reaction was set to run for 55 hours. Unlike many other Lactobacillus strains, L. pentosus is

Concentrations of different inhibitor compounds in WEH before and after membrane ﬁltration. Inhibitor compound Concentration, g/L

TABLE 1

Before

After

Acetic acid

38.86

2.17

Formic acid HMF

N/A 0.27

N/A N/A

Furfural

3.12

N/A



TABLE 2

SO present in the evaporator condensate

2 Hemicellulose of prepared fermentation media. might sugar reducecomposition the efficiency of the bio-

Medium

WEH, mL

V, mL

Xylose, g/L

methane production To address Mannose, Glucose, process. Galactose, Arabinose, this problem, the authors have recently g/L g/L g/L g/L

Rhamnose, g/L

Total sugars, g/L

med1 med2 med3 med4

300 400 500 600

500 400 300 200

36.90 41.22 64.15 86.34

6.22 5.75 2.04 1.73 sulphite mill evaporator condensates. Pilot 6.91 6.33 3.24 1.86 trial results on the use of this technol10.47 9.62 3.83 2.54 ogy indicated that 50%–93% of the SO 2 15.54 12.53 6.98 4.14 can be removed, depending on operating

1.44 1.91 3.48 3.98

54.08 61.47 94.10 129.50

used HFC technology to remove SO2 from

Fig. 6 - Sugar utilization and acid production profiles during fermentation of med2: (●) Lactic acid, (●) Acetic acid, (▼) Galactose, (▲) Arabinose, (▄) Glucose, (■) Xylose, ( ) Mannose, ( ) Rhamnose.


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capable of metabolizing both five-carbon and six-carbon sugars. Therefore, all sugars in the fermentation media were potential carbon sources for microorganism growth and lactic acid production. Acetic acid was produced as the main by-product. As shown in Fig. 6, during fermentation of med2, arabinose, glucose, rhamnose, and galactose were utilized within the first eight hours, while utilization of mannose and xylose started after eight hours. Mannose was consumed in the next 12 hours, and xylose was utilized only slowly. In the fermentation of med4 (Fig. 7), arabinose, glucose, rhamnose, and galactose were utilized within the first 24 hours, while utilization of mannose and xylose started after 10 hours. In this case, mannose was consumed in the next 14 hours, and xylose was utilized only slowly. After 55 hours of fermentation, all sugars contained in med2 were completely consumed, while in the case of med4, 118.11 g/L total sugars were consumed, and 10.39 g/L xylose remained unconsumed. The highest lactic acid concentration obtained was 43.66 g/L after 55 hours’ fermentation of med2, with a productivity of 0.99 g/(L.h) and a product yield (YP/S) of 0.72 g lactic acid /g total sugar consumed. Acetic acid (21.60 g/L) was produced as the main by-product. Figures 6 to 9 show that the production of acetic acid became pronounced after glucose had been exhausted. In the fermentation of med4 (Fig.7), 59.62 g/L lactic acid was produced after 55 hours of fermentation, with a productivity of 1.36 g/(L.h) and a product yield (YP/S) of 0.50 g-lactic acid /g- total sugar consumed. The concentration of the acetic acid produced was 21.13 g/L. During fermentation of med2 and


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med4, both five-carbon and six-carbon sugars were simultaneously consumed by L. pentosus. These results agree with those obtained by Bustos and coworkers [19], who reported that L. pentosus is a heterofermentative bacterium which utilizes glucose (a six-carbon sugar) through the EMP pathway and that in the absence of glucose, xylose (a five-carbon sugar) is utilized through the PK pathway. Conversion of hexoses and pentoses by L. pentosus has been predicted to obey the following overall stoichiometric equations [24]:

During this process, carbon dioxide, lactate, and acetate or ethanol are produced in varying proportions, depending on several factors such as aeration, initial sugar concentration, and even the presence of other proton and electron acceptors [19,25]. L. pentosus catabolizes one mole of glucose (or other six-carbon sugar) to yield two moles of pyruvate through the EMP pathway [19,21] (Fig. 1). The terminal electron acceptor in this pathway is pyruvate, which reduces to lactic acid. In the PK pathway, the pentose sugar undergoes oxidative reactions to generate one mole of each of glyceraldehyde 3-phosphate (GAP) and acetyl phosphate (Fig. 2). GAP is metabolized into lactic acid through the EMP pathway [21]. The acetylphosphate has two possible destinations, depending on environmental conditions. Under one condition, this molecule successively reduces into ethanol via acetyl-CoA and acetaldehyde intermediates. Under another condition, acetyl phosphate can produce acetate (acetic acid) through the enzyme acetate kinase. However, in the present study, no ethanol was detected. The fermentation profiles shown in Fig. 6 indicate that acetic-acid production started after all the arabinose, galactose, rhamnose, and glucose had been depleted, indicating that acetic-acid coproduction arose only from xylose and mannose consumption. Fermentation of med1 (Fig. 8) and med3 (Fig. 9) followed the same trend. The results obtained

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are in agreement with those reported in [19], working with the same bacterial strain (L. pentosus) but with a different raw material (vine-shoot trimmings hydrolysate). Although Bustos and coworkers [19] did not measure mannose, rhamnose, or galactose sugars, lactic acid and acetic acid production followed the same trend. When glucose is present during

the initial stage of fermentation, the sole product remains lactic acid while five-carbon (arabinose), six-carbon (glucose and galactose), and deoxy six-carbon (rhamnose) sugars are catabolyzed. Therefore, when glucose is present, one may infer that:




SPECIAL BIOREFINERY ISSUE Because of apparent acetic-acid production and the absence of ethanol production from the mixture of mannose and xylose at the end stage of fermentation, one may infer that the six-carbon sugars follow the EMP pathway while the fivecarbon sugars follow PK pathways when glucose is not present. In both med2 and med4, L. pentosus preferred to utilize arabinose (five-carbon), glucose (six-carbon), rhamnose (sixcarbon deoxy), and galactose (six-carbon) sugars first, before mannose (six-carbon) and xylose (five-carbon). In the case of med2, mannose and xylose utilization started after complete utilization of other sugars, while in med4, mannose and xylose utilization started after 10 hours of fermentation. Fermentation of med4 with higher initial glucose concentration (12.53 g/L) yielded a higher average rate of sugar utilization (2.68 g.L-1.h-1) than fermentation of med2 with a lower initial glucose concentration (6.33 g/L), which yielded a sugar utilization rate of 1.37 g.L-1.h-1. Compared to glucose, the conversion of xylose requires additional enzymatic steps. Some enzymes are inducible; therefore, there is a lag time before the enzymes required for assimilation appear when cells are exposed to xylose [26]. It has also been proposed that an increase in xylose concentration will result in increased intracellular concentrations of intermediates such as fructose 1,6-diphosphate, which inhibits enzyme activity during xylose metabolism [27]. Nevertheless, the ability of L. pentosus to utilize five-carbon and six-carbon sugar mixtures simultaneously makes it a potential microorganism for use in industrial fermentation of wood-extract hydrolysate. Effect of Sugar Loading on Lactic Acid Production

The effect of sugar loading on the production of lactic acid was studied (Fig. 10). Fermentation media containing 54.08 g/L sugars (med1), 61.47 g/L sugars (med2), 94.10 g/L sugars (med3), and 129.50 g/L sugars (med4) were used. As shown in Fig. 7, the final lactic

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Fig. 10 - Effect of initial sugar concentrations in wood hydrolysate-based medium on lactic acid production by L. pentosus.

acid concentration increased with an increase in sugar concentration in the fermentation medium. The maximum lactic acid concentration (65 g/L) was obtained after 106 hours of fermentation of med4. However, the yield was higher with fermentation media containing lower concentrations of WEH. The med1 medium had the highest product yield, 0.83 g-lactic acid/g-sugar, followed by med2 (0.72 g/g), med3 (0.65 g/g), and med4 (0.50 g/g). In addition, the time required for complete fermentation (or complete substrate utilization) generally increased with an increase in hydrolysate loading. Fermentation media (med1 and med2) which contained lower WEH concentrations required a shorter fermentation time to attain maximum lactic acid production than media with higher concentrations (med3 and med4). The low lactic-acid yields at higher WEH loading could be attributed largely to product inhibition because known microbial inhibitor compounds (furfural, HMF, and acetic acid) had been removed from WEH during the nano-filtration membrane process.

This result is in good agreement with reports in the literature [19,28] that product inhibition leads to decreases in fermentation rate and in microbial growth rate. In all fermentations, acetic acid was produced as the main by-product. Aceticacid production followed the same trend as lactic-acid production. Although there was no significant difference in the aceticacid production for media containing different levels of WEH, generally acetic acid concentration increased with an increase in sugar concentration in the fermentation medium. The med4 fermentation medium produced the highest acetic-acid concentration, 23.90 g/L, followed by med3 (22.61 g/L), med2 (21.61 g/L), and med1 (18.47 g/L). Acetic acid was produced mainly when mannose and xylose were the only remaining fermentable sugars. This observation indicates that in the presence of glucose, L. pentosus metabolizes both five-and six-carbon sugars to produce lactic acid exclusively, whereas in the absence of glucose, it metabolizes five-carbon sugars through the PK pathway to produce both lactic acid and acetic acid.


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Adaptation of L. pentosus to WEH for Lactic Acid Production

The effect of adaptation of seed-culture cells to concentrated WEH was studied. Generally, fermentation of the adapted L. pentosus strain on WEH-containing media showed higher lactic-acid yields and higher productivity than WEH fermentations performed with a native L. pentosus strain (Table 3). The adapted L. pentosus strain also required less time to achieve maximum lactic-acid production than the native strain. With the adapted L. pentosus strain, batch fermentation of med2 (containing 50% WEH (v/v)) led to a 15.5% increase in lactic-acid production, compared to a 2.0% increase in lactic-acid production in med4 (containing 75% WEH (v/v)). These results show that even with a WEH-adapted L. pentosus strain, fermentation of lactic acid from a medium containing high levels of WEH is affected by product inhibition, which is in agreement with the observations made earlier in this paper. Although wood-extract hydrolysate contains high concentrations of mixed sugars including xylose, mannose, glucose, arabinose, and rhamnose, little research has been reported in which woody biomass has been used as a carbon source for lactic-acid production. In the present study, batch fermentation of different WEHcontaining media led to high lactic-acid production levels ranging between 43.2 g/L and 65.02 g/L and representing 0.83 g/g and 0.54 g/g product yield respectively. The lactic-acid production and yield values obtained are higher than most values that have been reported in the literature. For example, Walton and co-workers [29] used the bacterial strain Bacillus coagulans MXL-9 to obtain 33 g/L lactic acid and a yield of 0.73 g/g from wood hemicellulose extracts; Bustos and coworkers [19] reported 46.0 g/L lactic-acid concentration and yield of 0.78 g/g from vine-trimming hydrolysate using a L. pentosus strain. Other research results that have been reported include lactic-acid production with a yield of 0.82 g/g from acid-treated softwood hydrolysates using L. casei subsp. Rhamnous strain [28] and lactic-acid prod-

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Fermentation of concentrated WEH using native and adapted Lactobacillus pentosus strains. Fermentation medium Parameter Strain med2 med4 Native Maximum lactic-acid 43.66 62.45 Adapted concentration, g/L 50.45 63.96 Native 55 75 Time required to attain maximum Adapted 45 52 lactic-acid production, h Native 0.72 0.50 Lactic-acid yield, g/g Adapted 0.78 0.54 Native 0.99 1.04 Lactic-acid productivity, g/(L×h) Adapted 1.15 1.53 TABLE 3

uction from wheat-straw hemicellulose hydrolysates at a concentration of 9 g/L (representing a yield of 0.90 g/g) using L. pentosus and 10 g/L (representing a yield of 0.61 g/g ) using L. brevis [30]. CONCLUSIONS

This study has demonstrated that batch fermentation of sugar maple wood-extract hydrolysate-based media using L. pentosus yields high productivity of lactic acid. The strain simultaneously utilized hexose and pentose sugars in the fermentation media. In the first 12 hours of fermentation, the strain preferably utilized arabinose, glucose, rhamnose, and galactose, while utilization of mannose and xylose started after 12 h, with mannose being consumed in the next 12 h. However, L. pentosus growth rate and product yield were inhibited by high concentrations of lactic acid produced during fermentation. Although use of an L. pentosus strain adapted to concentrated WEH led to significant increases in lacticacid productivity and yield for fermentation media containing lower concentrations of WEH, there was no significant effect on product yields with fermentation media containing high WEH concentrations. The low lactic-acid yield could be attributed largely to product inhibition because known microbial inhibitors contained in wood hydrolysate had been removed by a nano-filtration process. The main byproduct, acetic acid, is produced after glucose has been depleted. Acetic-acid production is minimal when glucose is present in the sugar mixture.

ACKNOWLEDGEMENTS

The authors are grateful for research support from the Biorefinery Research Institute and the Department of Paper and Bioprocess Engineering. Thanks also to Dr. Thomas E. Amidon, Mr. Dave Kiemle, and Mr. Christopher D. Wood for their help with these studies. REFERENCES 1.

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