Cellulase production by continuous culture of Trichoderma reesei Rut C30 using acid hydrolysate prepared to retain more oligosaccharides for induction

Bioresource Technology 101 (2010) 717–723

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Cellulase production by continuous culture of Trichoderma reesei Rut C30 using acid hydrolysate prepared to retain more oligosaccharides for induction Chi-Ming Lo, Qin Zhang, Nicholas V. Callow, Lu-Kwang Ju * Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906, USA

a r t i c l e

i n f o

Article history: Received 18 February 2008 Received in revised form 17 July 2009 Accepted 12 August 2009 Available online 22 September 2009 Keywords: Acid hydrolysis Cellulase Detoxification Air stripping Trichoderma reesei

a b s t r a c t An acid hydrolysate was prepared by a procedure chosen for retaining more oligosaccharides to improve the cellulase-inducing capability when used as substrate in the fungal fermentation for cellulase production. The effect was evaluated with continuous culture of Trichoderma reesei Rut C30 at the dilution rates of 0.03–0.08 h1. The specific cellulase production rates were found to be relatively constant at 8.9 ± 0.3 (FPU/g dry cells-h), except for the lower rate, i.e., 7.2 (FPU/g-h), at the lowest dilution rate investigated (0.03 h1). The former value was slightly higher than the rate obtained with a lactose-based medium, i.e., 8.2 (FPU/g-h). The maximum specific cell growth rate supported by the hydrolysate-based medium was 0.096 (h1) and the apparent cell yield increased from 0.44 to 0.57 (g dry cells)/(g consumed reducing sugars) with increasing dilution rates. The best-fit maximum/ideal cell yield (without endogenous metabolism) was 0.68 (g/g), the endogenous substrate consumption rate was 0.023 (g reducing sugars)/(g dry cells-h), and the specific cell death rate was 0.016 h1. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Cellulase is a group of enzymes that collectively have the capability of catalyzing the hydrolysis of cellulose to glucose (Ladisch et al., 1981). The glucose produced can then be used as the substrate for producing many fermentation products including ethanol (McMillan, 1994). Effective cellulase production is therefore critically important to biorefinery, to utilize renewable lignocellulosic materials for production of fuel and chemicals (Araujo and D’Souza, 1980; Brandt et al., 1973). Cellulase is industrially produced by fermentation processes using Trichoderma reesei, the anamorph of the fungus Hypocrea jecorina (Mohagheghi et al., 1988; Shin et al., 2000). The fungal cellulase synthesis is regulated by both induction and end-product (glucose) repression (Ilmen et al., 1997). T. reesei Rut C30 is a commonly used strain known for its higher resistance to glucose repression (Montenecourt and Eveleigh, 1979). Nonetheless, our studies with this strain in continuous culture studies indicated still appreciable glucose repression on cellulase synthesis (Ju and Afolabi, 1999; Lo, 2008). In the most recent continuous culture study with lactose or mixed lactose and glycerol as the substrate(s), the repression constant of glucose r;FPU ) on cellulase synthesis was found to be about 0.07 g/L when (K Glu the repression was modeled with the simple ‘‘uncompetitive” mechanism (Lo, 2008):

* Corresponding author. Tel.: +1 330 972 7252; fax: +1 330 972 5856. E-mail address: [email protected] (L.-K. Ju). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.08.056

SRFPU ¼ bFPU

L   K FPU þ L 1 þ KGlu r;FPU L Glu

where SRFPU is the specific production rate of cellulase (assayed as the Filter Paper Units, FPU) (FPU/g dry cells-h), bFPU is the maximum rate under optimal induction (by lactose) and no repression (by glucose), L and Glu are lactose and glucose concentrations (g/L), is the half-saturation lactose concentration for induction and K FPU L (g/L). The oligosaccharides formed during the cellulose hydrolysis are believed to play important roles in the natural cellulase induction (Huang, 1975; Ladisch et al., 1981). Solid cellulosic materials have therefore often been used as both the substrate and the source of inducers in the fermentation processes for cellulase production (Huang, 1975; Lee and Fan, 1982, 1983). Solid substrates are, however, hard to handle, particularly if continuous or periodic feeding and broth removal are desired. The presence of solids can also increase the burden on agitation and decrease the oxygen transfer efficiency of the bioreactors. Lower solid concentrations have been shown to yield higher cellulase production (Szengyel et al., 1997). An attractive alternative is to use media prepared with the cellulose hydrolysates that contain high enough contents of oligosaccharides to provide adequate induction. In a previous study, the hydrolysates prepared from hardwood sawdust, using a two-stage acid hydrolysis process developed by Tennessee Valley Authority (Muscle Shoals, AL), were investigated in batch fermentation of T. reesei Rut C30 (Lo et al., 2005). The

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hydrolysates were prepared with different boiling durations, ranging from 15 to 120 min, in the second-stage hydrolysis. (More description is given in the Section 2.) All of the examined hydrolysates supported cell growth, although with longer lag phases than those observed in the controls with media of mixed glucose and Avicel (Sigma) cellulose. The longer lag phases were attributed to the presence of some inhibitory byproducts commonly found in acid hydrolysates (Larsson et al., 1999; McMillan, 1994; Palmqvist et al., 1997). While all of the hydrolysates also induced cellulase production, the hydrolysates prepared with the shortest boiling durations, i.e., 15 and 30 min, gave the highest cellulase productivity (Lo et al., 2005). The higher inducing potency of these hydrolysates corresponded well with the higher oligosaccharide contents measured in these hydrolysates. Cellulase synthesis by T. reesei Rut C30 is largely, if not completely, non-growth-associated (Velkovska et al., 1997). Batch fermentation is not the best way of evaluating the inducing ability of the hydrolysate because much of the oligosaccharides may have been hydrolyzed before the cell concentration is high enough to effect more substantial cellulase production. A continuous culture study was therefore conducted in this work. The examined hydrolysate was prepared with only 15 min of boiling in the secondstage of acid hydrolysis.

2. Methods T. reesei Rut C30 (NRRL 11460) was used in this study. Detailed descriptions for culture maintenance, hydrolysate preparation, medium composition, and analytical methods are available elsewhere (Lo et al., 2005; Zhang et al., 2007). Only brief descriptions are given here. 2.1. Hydrolysate preparation The sawdust was first mixed for 30 min with 80% (w/w) sulfuric acid at about 1:1 solid to acid ratio (by wt), to form a viscous ‘‘gel”. The gel was then diluted and mixed for 15 min with additional water. In the previous study, the amount of water added was 3 times the volume of concentrated acid used initially (Lo et al., 2005); in this study, the amount was reduced by half in order to increase the hydrolysate sugar concentration for supporting higher cell concentrations in the continuous culture. (In the previous batch study, the maximum cell dry-weight concentrations (CDW) were only about 3 g/L (Lo et al., 2005).) The diluted slurry was filtered (20-mesh, 850-lm opening) to remove the unreacted solids. The filtrate was then boiled gently for 15 min, with water addition to compensate for vaporization loss. After being quickly cooled in ice/water bath, the hydrolysate was over-limed with Ca(OH)2 to pH 9 and centrifuged to remove the precipitates formed during over-liming. The supernatant collected was adjusted to pH 7 and used to prepare the feed medium for the continuous culture study. The neutralized hydrolysate contained about 20 g/L of total reducing sugars, including 4.6 g/L of glucose. 2.2. Medium The medium composition was essentially the same as that of (Mandels et al., 1976) for 10 g/L of carbon source (cellulose, in their case). Hydrolysate, added with measured amounts of deionized water and peptone, was autoclaved separately from a solution containing all other nutrients at 20-fold concentrations. The two autoclaved solutions were cooled and then mixed inside a laminar flow hood. The concentration of total reducing sugars in the feed medium thus prepared was measured to be 13.5 ± 0.1 g/L.

2.3. Continuous culture The continuous culture study was carried out in a bioreactor (Newbrunswick BioFlo 110) with 1 L of medium. The culture conditions were controlled at pH 5.0 ± 0.2 (by addition of 2 N NaOH), dissolved oxygen concentrations (DO) = 70%–85% air saturation (by adjustment of the oxygen level in the influent gas, using the LabView 7.1 software from National Instruments (Austin, TX), agitation speed = 500 rpm, and temperature = 25 ± 1 °C. Olive oil was added automatically as the anti-foam. The dilution rate was studied in the range of 0.03–0.08 h1. Continuous feeding was started after the initial batch culture had reached the early stationary phase. To ensure that the culture reached the steady state, periodical samples were taken after the culture had been maintained at the specific dilution rate for at least 5 residence times. The concentrations of intracellular proteins and reducing sugars as well as the cellulase activity were measured. The results reported were the averages of at least 3 samples. The broth was removed periodically from the bioreactor by a computer-controlled Peristaltic pump, every 15 min at the dilution rates <0.06 h1 and every 10 min at higher dilution rates. The periodical removal was chosen over the simple overflow arrangement because the fungal broth was viscous and, with the low flow rates involved in the laboratory-scale operation, the mycelial cells tended to settle and/or attach to the tubing wall. As these cells grew in the tubing, they formed the flow restriction sites for further cell accumulation. As a result of the combined cell growth and accumulation, the tubing was occasionally clogged in our earlier experiments using the overflow approach. Also, the overflow medium tended to be depleted in mycelial concentration, leading to artificial cell retention in the bioreactor. The computer-controlled periodical removal enabled the pumping at much higher rates during a short period and eliminated the above problems. A mathematical model simulation of the above operation of periodical broth removal was done by taking into account of the increase in broth volume and changes in cell and substrate concentrations during the period between two consecutive removals, as well as the step decrease in broth volume by each removal. The simulation results indicated that the average behaviors of thus operated systems would be essentially the same as those of ideal continuous culture. The model simulation and the simulated results are presented in the Discussion section. 2.4. Analytical methods The samples taken from the bioreactor were centrifuged (at 14,500g for 10 min) to separate the cells from the supernatant. The cells were washed twice with deionized water before being used to determine the intracellular protein concentration. The supernatant collected was analyzed for the reducing sugar concentration and the cellulase activity. Detailed descriptions of the methods are available elsewhere (Lo et al., 2005; Wu and Ju, 1998; Zhang et al., 2007). Briefly, the reducing sugar concentration was measured by the non-specific dinitrosalicylic acid (DNS) method, based on the color formation of DNS reagent when heated with reducing sugars. The intracellular protein concentration was determined by using the Bradford protein assay kit (Sigma–Aldrich #B6916), based on the binding of Coomassie Brilliant Blue G-250 dye with the proteins containing basic and aromatic amino acid residues. The cell dry-weight concentration was estimated from the measured intracellular protein concentration using a preestablished correlation: [cell dry-weight concentration] (g/ L) = [intracellular protein concentration] (g/L)  8.0 (±0.5). The total cellulase activity, in Filter Paper Unit (FPU), was measured by the standard filter paper assay (Afolabi, 1997).

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3. Results The results of cell concentration, cellulase FPU activity, and residual reducing sugar concentration obtained in the continuous culture study at the dilution rate (D) of 0.03–0.08 h1 are shown in Fig. 1.

The best-fit value for l (=0.096 h1) agreed well with the previous results of batch fermentation (Lo et al., 2005) where l was observed to be about 0.1 h1. This growth rate was lower than that obtained with glucose as the substrate (0.13 h1) (Lo, 2008), consistent with the observation of some inhibitory effects of the hydrolysate. Nonetheless, the cell growth rate on the hydrolysate was still appreciably higher than that on galactose (0.06 h1) (Lo, 2008).

3.1. Reducing sugar concentration 3.2. Cell concentration

l¼

lS ¼ D þ kd K þS

ð1Þ

where l is the specific cell growth rate (h1), l is the maximum specific cell growth rate, K is the Monod (half-maximum) constant for the substrate (i.e., the ‘‘degradable” portion of the reducing sugars), and kd is the specific cell death rate (h1). Eq. (1) can be rearranged to

S¼

KðD þ kd Þ l  D  kd

ð2Þ

Specific Celluase Production Rate (FPU/g-h)

Cell or Reducing Sugars Concentration (g/L)

The value of kd was determined to be 0.016 h1, as described in Section 3.2. With Eq. (2) and the experimental results of S at different D, the values for l and K were estimated to be 0.096 h1 and 0.00057 g/L, respectively, using the built-in Solver program in Microsoft Excel to minimize the sum of square of the differences between the experimental and calculated values for S.

The cell dry-weight concentrations (CDW) increased slightly as D increased from 0.03 h1 to 0.05 h1 and then remained relatively constant at 6.64 ± 0.19 g/L at higher D. (The p value was 2.0  104 when all data were included in the ANOVA analysis.) Similarly, the ‘‘apparent” cell yield (Y AP X=S ) from the (degradable) hydrolysate sugars increased from 44% (i.e., 0.44 g CDW/g consumed reducing sugars) at D = 0.03 h1 to 49% at D = 0.042 h1 and eventually to 57% at D = 0.08 h1. As shown in Table 1, these apparent cell yields, 0.44– 0.57, compared well with those reported in the literature for T. reesei on glucose or lactose, mostly from continuous culture studies. The change of apparent cell yield with the dilution rate, observed in this study, indicated relatively substantial endogenous metabolism (or cell death) when the cells were grown in the hydrolysate-based medium. This observation is consistent with the finding in the previous batch fermentation study (Lo et al., 2005) that the growth of T. reesei Rut C30 in the hydrolysate-based medium required much longer lag phases. The maintenance coefficient (i.e., specific substrate consumption rate by endogenous metabolism), ms, can be determined using the following equation (Shuler and Kargi, 1992):

1 Y AP X=S

¼

1 YM X=S

þ

ms D

ð3Þ

where Y M X=S is the maximum cell yield (without endogenous metabolism or maintenance energy) and ms can be related to the cell death rate kd (h1) by the following equation:

ms ¼

kd YM X=S

ð4Þ

:

The values of 1=Y AP X=S were therefore plotted against (1/D) in Fig. 2. Accordingly, ms was determined to be 0.023 g sugars/g CDW-h, 1 . YM X=S was 67.6%, and kd was about 0.016 h

10

1400

8

1200

6

1000

4

2

0 0.02

800

Cells Reducing Sugars Cellulase SRFPU

Cellulase (FPU/L)

The residual reducing sugar concentrations were constant, at 1.18 (±0.01) g/L, at all but the highest D (0.08 h1) examined. The residual sugar concentration was slightly higher, 1.46 g/L, at D = 0.08 h1. (The single-factor Analysis of Variance, ANOVA, gave a p value of 0.95, >>0.05, when only the data at D < 0.08 h1 were analyzed and a p value of 5.3  105, <<0.05, when all data were included in the analysis. The ANOVA results indicated that the effect of D on the reducing sugar concentrations was insignificant at D < 0.08 h1 but significant at the higher D, 0.08 h1.) The non-zero reducing sugar concentrations at low D indicated that some of the ‘‘reducing sugars” in the acid hydrolysate, detected by the DNS method, were not consumable by T. reesei. With 13.5 g/L of total reducing sugars in the feed, the above value corresponded to about 8.8% non-consumable reducing sugars in the acid hydrolysate. Following the simple unstructured model for continuous culture (Shuler and Kargi, 1992), at steady state,

600

400 0.04

0.06

0.08

D (h-1) Fig. 1. Profiles observed in the continuous culture of T. reesei Rut C30 with the hydrolysate-based medium.

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Table 1 Comparison of apparent cell yields reported for continuous culture of T. reesei. Substrate

YX/S

Glucose

0.63 0.45 0.53 0.74 0.58 0.63 0.44 0.60 0.44–0.57

Lactose

Sawdust hydrolysate

*

T. reesei strain

Culture conditions *

Rut C30 QM9414 T. viride QM 9123 Trichoderma sp. C5 C5 MCG 77 Rut C30 Rut C30

28 °C 28 °C* 30 °C, pH 4.0 30 °C, pH 4.0 30 °C, pH 5.0 30 °C, pH 5.0 32 °C (two-stage) 30 °C, pH 4.8 25 °C, pH 5.0 DO > 70%

Reference Seidl et al. (2008) Seidl et al. (2008) Brown and Zainudeen (1977) Nagai et al. (1976) Chaudhuri and Sahai (1994) Chaudhuri and Sahai (1993) Ryu et al. (1979) Pakula et al. (2005) This work

These were from batch fermentation carried out in shake flasks at 200 rpm, included to show the cell yields of different strains.

2.4

d ðVC i Þ ¼ FC 0i þ Vr i dt

1/YX/SAP

2.2 2 1.8

y = 0.023x + 1.480 R2 = 0.967

1.6 1.4 0

5

10

15

20

25

30

35

1/D (h) M Fig. 2. Plotting of 1=Y AP X=S against (1/D) for estimation of maximum cell yield (Y X=S ) and maintenance coefficient (ms).

3.3. Cellulase activity As the dilution rate was increased from 0.03 to 0.08 h1, the cellulase (FPU) activity declined almost linearly from 1320 FPU/L to 760 FPU/L (Fig. 1) (p = 1.2  1010). On the other hand, the specific cellulase production rates SRFPU were relatively constant at 8.9 ± 0.3 (FPU/g CDW-h) for D = 0.042–0.08 h1, although lower at 7.2 (FPU/g CDW-h) for D = 0.03 h1. The decrease of FPU with increasing D was therefore not because of slower FPU production; instead, it was because of the faster dilution (and removal) at higher D. A similar FPU profile was observed in another continuous culture study recently conducted in this laboratory, using lactose as the inducing substrate (Lo, 2008). The maximum FPU observed in that study was 1200 FPU/L and the specific FPU production rate was also approximately constant (except at the lower D, similar to this study), at about 8.2 (FPU/g CDW-h). On the other hand, in an earlier continuous culture study using a wastepaper hydrolysate prepared with a different procedure (including acid pretreatment and 4-day enzymatic hydrolysis), the specific FPU production rate decreased significantly from 12.2 (FPU/g CDW-h) at D = 0.12 h1 to 2.2 (FPU/g CDW-h) at D = 0.012 h1 (Ju and Afolabi, 1999). The much lower SRFPU found at lower D was attributed to insufficient residual inducing oligomers, due to more thorough degradation in these systems with longer retention times. Given the observations of these other two studies, the finding of relatively constant SRFPU in the current study implied that the acid hydrolysate prepared by the particular procedure indeed retained more (and, perhaps, sufficient) oligosaccharides for inducing cellulase production in the continuous culture of T. reesei. 4. Discussion 4.1. Model simulation of frequent but non-continuous broth removal For any component i, the material balance made during the period between two consecutive broth removals is

ð5Þ

where, t is time (h), with t = 0 corresponding to the moment after the first removal and t = s to the moment right before the second removal (thus, s represents the interval between removals); V is the medium volume (L) at the instant t; Ci is the concentration (g/ L) of component i (which is cell or reducing sugar concentration in this model simulation; cellulase activity, although possible, is not simulated here) and C 0i is the concentration in the fresh feed (which is 0 g/L for cell concentration and 13.5 g/L for reducing sugar concentration, respectively); F is the medium feed rate (L/h); and ri is the volumetric rate of generation (g/L-h) for component i. Due to the feeding, V increases with t from V0 (at t = 0) to Vs (at t = s), according to the following equation

V ¼ V 0 þ Ft ¼ V av g þ Fðt  s=2Þ: av g

0

ð6Þ

s

where V ¼ ðV þ V Þ=2, representing the average medium volume during the interval. In addition, the periodical broth removal was to achieve an equivalent dilution rate, i.e.,

D¼

F : V av g

ð7Þ

Eq. (5) can be rearranged into the following form, after substituting V and F with Eqs. (6) and (7):

dC i D ¼ ri þ ðC 0  C i Þ 1  Dðs=2Þ þ Dt i dt

ð8Þ

ri in Eq. (8) can be described by the following equations according to the simple unstructured model,

for cell concentrationðXÞ ri ¼ ðl  kd ÞX for substrateðreducing sugarÞconcentrationðSÞ r i ¼ 

ð9Þ

lX YM X=S

ð10Þ

where l ¼ lS=K þ S as in Eq. (1). Substituting Eqs. (9) and (10) into Eq. (8) gives two simultaneous ordinary differential equations (ODEs): one for dX/dt, the other for dS/dt. The two ODEs and the Monod equation were coded in Excel’s Visual Basics for Applications, with the two ODEs being solved numerically using the fourth-order Runge–Kutta method (Liengme, 2002). Using the kinetic parameters reported earlier in the Results section, i.e., l = 0.096 h1, K = 0.00057 g/L, Y M X=S = 0.676, and kd = 0.016 h1, the profiles of cell and reducing sugar concentrations could be simulated for the systems with frequent periodical broth removal. As an example, the simulated results for the system with D = 0.05 h1 and s = 0.25 h (i.e., 15 min) are shown in Fig. 3 for two cycles of broth removal. The results expected from ‘‘true” continuous culture are also shown in Fig. 3 for comparison. The simulation clearly demonstrated that the operation of frequent broth removal can achieve essentially the

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1.18642

6.33664 6.33662

Cell

1.18638

6.33660

1.18636

6.33658

Periodical removal

Continuous culture

1.18634

6.33656

1.18632

6.33654

1.18630

6.33652

1.18628

Cell (g CDW/L)

Reducing Sugars (g/L)

1.18640

6.33650

Reducing Sugars

1.18626

6.33648 0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Time (h) Fig. 3. Comparison of simulated cell and reducing sugar concentrations between ‘‘ideal” continuous culture and the ‘‘real” system with broth removal every 15 min (for 2 cycles) at the (equivalent) dilution rate of 0.05 h1.

same outcomes (on average, very slight over-approximation in reducing sugar concentrations and under-approximation in cell concentrations) as the ideal continuous culture while eliminating the practical difficulties associated with the use of simple overflow for continuous culture of mycelial fungi.

Table 2 Colony growth of T. reesei Rut C30 on agar plates with acid hydrolysate of hardwood sawdust. (The hydrolysate had been acidified to pH 5.0 and aerated for different durations before being used to prepare the agar plates at different hydrolysate concentrations.). Duration of aeration for inhibitor removal (days)

4.2. Inhibitory effects and air-stripping detoxification of acid hydrolysate The extent of inhibition exerted by the acid hydrolysate was found to be rather sensitive to the hydrolysate preparation procedure. The previous batch fermentation was conducted with the hydrolysates prepared in a same batch (Lo et al., 2005). The continuous culture study reported in the Results section was carried out with another batch of hydrolysate. Yet another batch of hydrolysate was prepared but not used in the continuous culture study because of the severe inhibition observed: the medium prepared from this hydrolysate failed to support any cell growth when used for pre-culturing inoculum, and the medium caused the cells to gradually turn to spores when added as the feed to a continuous culture originally maintained on a lactose-based medium. An experiment was made to investigate if the inhibitory effects could be removed or reduced by simply bubbling filter-sterilized air through pre-acidified hydrolysate (pH 5). After 3, 5, and 7 days of aeration, the hydrolysate was used to prepare agar plates containing 39 g/L of potato dextrose agar and 50%, 75% and 100% of hydrolysate, respectively. (Note that the 100% hydrolysate had about 20 g/L reducing sugars.) Duplicate plates were prepared for each combination of aeration duration (3, 5, or 7 days) and hydrolysate strength (50%, 75% or 100%). After spreading 1 ml of inoculum on each plate, the cell growth, indicated as colony formation, was monitored. The results obtained are shown in Table 2. Clearly, aerating the acidified hydrolysate helped to reduce the inhibitory effects to the fungal cells. This simple experiment suggested the feasibility of using the air stripping method for potential detoxification of the acid hydrolysate. Acetic acid is a main inhibitory compound in acid hydrolysate (Szengyel and Zacchi, 2000). Although a well known Volatile Fatty Acid, acetic acid has a rather low vapor pressure, e.g., 15.8 mm Hg at 25 °C (Lange, 1992). To investigate if the reduced inhibition of aerated hydrolysate could be at least partially caused by the removal of acetic acid, the following experiment was made with acetic acid solutions at 1 g/L and 10 g/L concentrations. One set of the solutions was adjusted to have an initial pH of 3.0, the other set to

3 5 7

Hydrolysate strength 50%

70%

100%

 + ++

 + +

  

Note: ‘‘”: no apparent growth; ‘‘+”: slow growth; ‘‘++”: moderate growth.

a pH of 6.0. (pKa of acetic acid is 4.8 at 25 °C.) Moist air, prepared by passing air through a humidification bottle and a liquid trap (to remove any oversaturated water), was filtered through a 0.2-lm filter (Whatman Polyvent, Piscataway, NJ) and then continuously bubbled through the acetic acid solutions. Periodic samples were taken and analyzed by reversed-phase high-performance liquid chromatography, using a Hewlett–Packard LC1100 series HPLC fitted with a G1315A diode array detector and a Waters YMC-ODS AQ12S052546WT column operating at 30 °C. The mobile phase was 20 mM sodium phosphate buffer, pH 2.8, at 0.7 ml/min. Despite the use of humidified air, some water loss occurred over the long experimental period (192 h). The final solution volume was measured to determine the rate of water loss in each system, after taking into account of the removed sample volumes. The total water loss ranged from 13%–24%. The water loss rate was assumed to be constant in each system, and the raw acetic acid concentrations were adjusted accordingly. The adjusted concentrations were shown in Fig. 4. As shown in the figure, the acetic acid concentrations in the solutions at pH 6 (>pKa) increased slightly over time, presumably due to the imperfect adjustment for water loss. On the other hand, the concentrations in the solutions at pH 3 (
14

1.4

12

1.2

10

1

8

0.8

6

0.6

4

0.4

2

0.2

Acetic Acid (g/L) (1 g/L Systems)

C.-M. Lo et al. / Bioresource Technology 101 (2010) 717–723

Acetic Acid (g/L) (10 g/L Systems)

722

0

0

0

50 10 g/ L, pH 3

100 150 Time (h) 10 g/ L, pH 6 1 g/ L, pH 3

200 1 g/ L, pH 6

Fig. 4. Changes of acetic acid concentration with time by bubbling moist air through 1 g/L and 10 g/L acetic acid solutions with initial pH of 3.0 or 6.0. The acetic acid concentrations shown had been adjusted for the water loss during the long aeration process.

5. Conclusions The hydrolysate prepared by the chosen acid hydrolysis procedure was shown to have slightly better cellulase-inducing potency than lactose, yielding 8.88 ± 0.27 (FPU/g dry cells-h) at D > 0.042 h1. The hydrolysate was also found to contain about 8.8% of nondegradable reducing sugars. The hydrolysate-based medium supported cell growth with a maximum specific growth rate of 0.096 h1 and apparent cell yields in the range of 44% to 57%. The maximum cell yield (without endogenous metabolism) was estimated to be 68%, the endogenous substrate consumption rate was 0.023 (g reducing sugars)/(g dry cells-h), and the specific cell death rate was 0.016 h1. The hydrolysate had some inhibitory effects to the cells, and the extent of inhibition appeared to be rather sensitive to slight differences in the hydrolysis procedure. A simple, yet to be optimized, operation of aerating the acidified hydrolysate was shown feasible to reduce the inhibitory effects, presumably due to partial removal of volatile compounds such as acetic acid. Acknowledgement This project was supported by Initiative for Future Agriculture and Food System Grant Number 2001-52104-11476 and by Biomass Research and Development Initiative Grant Number 683A75-7-610, both from the USDA Cooperative State Research, Education, and Extension Service. The authors are very grateful for the help of Dr. Patrick Lee at Tennessee Valley Authority (Muscle Shoals, AL) in preparing the acid hydrolysate for the study. References Afolabi, O.A., 1997. Wastepaper Hydrolysate as Substrate and Inducer for Cellulase Production. Department of Chemical Engineering. The University of Akron, Akron, OH. pp. 77–85. Araujo, A., D’Souza, J., 1980. Production of biomass from enzymic hydrolysate of agricultural waste. Journal of Fermentation Technology 58, 399–401. Brandt, D., Hontz, L., Mandels, M., 1973. Engineering aspects of the enzymic conversion of waste cellulose to glucose. AIChE Symposium Series 69, 127–133. Brown, D.E., Zainudeen, M.A., 1977. Growth kinetics and cellulase biosynthesis in the continuous culture of Trichoderma viride. Biotechnology and Bioengineering 19, 941–958. Chaudhuri, B.K., Sahai, V., 1993. Production of cellulase enzyme from lactose in batch and continuous cultures by a partially constitutive strain of Trichoderma reesei. Enzyme and Microbial Technology 15, 513–518. Chaudhuri, B.K., Sahai, V., 1994. Comparison of cellulase growth and maintenance parameters for cellulase biosynthesis by Trichoderma reesei C5 with some published data. Enzyme and Microbial Technology 16, 1079–1083.

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