Lactic acid from wood

Process Biochemistry Vol. 31, No. 3, pp. 271-280, 1996 Copyright @ 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 S 15.00 + 0.00 ELSEVIER

0032-9592(95)00059-3

Lactic Acid from Wood I. C. Paraj6,* J. L. Alonso & V. Santos )epartment of ChemicalEngineering,Universityof Vigo(CampusOrense),Las Lagunas,32004 Orense, Spain Received 12 May 1995;accepted2 July 1995)

Pinus pinaster orEucalyptus globulus wood samples were subjected to chemical processing (alkaline extraction or delignification-swelling treatments) under ~elected conditions and the solid residues produced used as substrates for the enzymic hydrolysis of their polysaccharide fractions. The sugar solutions obtained were supplemented with nutrients and used as fermentation media for lactic acid production with Lactobacillus delbrueckii NRRL B-445. The kinetics of the bioconversion stage were modelled, and the suitability of hydrolysates for fermentation purposes were discussed.

INTRODUCTION

solutions by bacteria, but recently the fungus Rhizopus oryzae was also proposed for lactic acid fermentation.8,9 Several carbon sources (including glucose, fructose, sucrose, lactose and starch) have been used for this purpose; and the modelling of the fermentation kinetics in batch or continuous cultures has been studied by several authors.6, 7,10 -21 Wood is an interesting raw material for chemical or biotechnological processes, owing to its renewable character, widespread location, large availability and low price. The polysaccharide fraction of wood can be converted into sugars in reaction media catalysed by acids or enzymes. The enzymatic saccharification of native wood proceeds slowly and leads to reduced reaction yields, but both the kinetics and polysaccharide conversion can be markedly improved by chemical pretreatment of substrates. After supplementation with nutrients, the sugar solutions obtained from enzymic hydrolysis of pretreated cellulosic substrates are potential fermentation media that can be used, for example, for ethanol production.

More than 50% of the lactic acid produced in the world (about 40000 ton/yr) is utilized in food technology as pH regulator, microbial preservative or buffering agent. In the chemical industry, lactic acid is used as solvent, for pH regulation, for polymer manufacture and other purposes including the possibility of making biocompatible or biodegradable polymers from lactic acid. 1-3 Moreover, ammonium lactate has been favourably compared as feed supplement with other non-proteic nitrogen s o u r c e s , 4-7 and both lactic acid and lactates are widely employed in the formulation of pharmaceutical products. 2 Lactic acid can be obtained by either chemical or biotechnological means. Chemical synthesis starts from acetaldehyde, which is reacted with I-/CN to give lactonitrile, this compound then being hydrolysed to lactic acid. 1 The biotechnological procedures for lactic acid production are traditionally based on the bioconversion of sugar "To whomcorrespondenceshouldbe addressed. 271

272

J.C. Paraj6et al.

Only a few studies have explored the possibility of producing lactic acid from lignocellulosic substrates. Abe and Takagi 22 explored the simultaneous saccharification and fermentation of cellulose powder and newspaper to lactic acid in media containing cellulases and Lactobacillus delbrueckii cells. In related studies, Takagi23 considered the inhibition of cellulases by lactic acid, and Chosson et al. 24 evaluated the inhibition of the hydrolytic ability of Cellulomonas caused by lactic acid. This work deals with the bioconversion of wood into lactic acid via enzymic hydrolysis. Pinus pinaster or Eucalyptus globulus wood samples were subjected to chemical treatments to render them susceptible to saccharification by cellulase-cellobiase mixtures. The solid residues from these treatments were used as hydrolysis substrates, and the resulting sugar solutions were supplemented with nutrients and used for the production of lactic acid in a subsequent fermentation step. The kinetics of the overall process was modelled (including generation of lactic acid and biomass, as well as substrate consumption), and the suitability of hydrolysates as fermentation media was evaluated.

MATERIALS AND METHODS Raw material Pinus pinaster and Eucalyptus globulus wood chips were obtained from local industries, milled to pass a 0"5 mm screen and air-dried. The milled samples were homogenized in separate lots for pine and eucalyptus, in order to avoid differences among aliquots of a given kind of wood, and stored in polyethylene bags.

Composition of wood Wood samples were subjected to quantitative sacchafification with 72% HESO4 following standard methods. 25 The solid residue was considered to be lignin. The liquors from quantitative saccharification were neutralized and analysed for glucose and total monosaccharides by HPLC as reported elsewhere. 26 The content of cellulose was calculated from the glucose concentration of hydrolysates and the total monosaccharide concentration was used to estimate the polysaccharide content of wood. The hemicellulose content was calculated as the difference between the polysaccharides and cellulose contents of samples.

Chemical processing of wood Wood samples were treated in batch reactors with selected chemical agents (NaOH, H O O C C H 3, NaC10 or NH4OH) under selected operational conditions (see below). The solid residues were washed, air-dried and subjected to quantitative saccharification (by the same method employed for raw wood). The composition of solid residues was characterized by the contents of lignin, total polysaccharides and cellulose. Enzymic hydrolysis Processed samples were used as substrates for hydrolysis experiments, which were carried out in stirred Erlenmeyer flasks at 48.5°C using commercial enzymes. Enzyme concentrates ('Celluclast' cellulases from Trichoderma reesei and 'Novozym' fl-glucosidase from Aspergillus niger) were a gift from Novo Nordisk Bioindustrial (Madrid, Spain). The operational conditions used for enzymic hydrolysis were selected according to previous studies (see below). Fermentation experiments Wood hydrolysates were supplemented with nutrients to produce a modified medium based on that proposed by Sharpe et al. 27 and used earlier for lactic fermentation. 6 The medium contained: yeast extract, 5 g/litre; peptone, 10 g/litre; sodium acetate, 5 g/litre; sodium citrate, 2 g/litre; K2HPO4, 2 g/litre; Tween 80, 1 ml/litre; MgSO4.7H20, 0-58 g/litre; MnSO4.H20 0.12 g/ litre; and FeSO4" 7H20, 0"05 g/litre. For comparative purposes, additional experiments were carried out with standard glucose solutions containing 20 or 50 g glucose/litre and the same type and concentration of nutrients of the fermentation media made from hydrolysates. Lactobacillus delbrueckii N R R L B-445 (obtained from the Northern Regional Research Center, USDA, Peoria, IL, USA) was used to perform the bioconversion of the carbon sources into lactic acid. The microorganism was cultured in an orbital shaker in the selected media for 15 h, and used as inocula for pH-controlled experiments. These assays were performed in a Biostat B batch fermentor at 42°C with a pH control set-point of 5.8, using 4 U NaOH solution for neutralization. The dilution effect caused by neutralization was considered in the formulation of material balances. During the experiments, the agitation speed was fixed at 200 rpm. At given fermentation times, samples were withdrawn from the media and cen-

Lactic acidfrom wood trifuged. The pellets were washed and used for the determination of dry biomass. The supernatants were immediately analysed for substrate and lactic acid by the same HPLC method used for sugars.

Correlation of data The experimental data obtained in fermentation trials were fitted to the proposed models by linear or nonliner regression using commercial software (TableCurve from Jandel Scientific, Corta Madera, CA, USA). RESULTS AND DISCUSSION

Composition and chemical processing of samples The chemical processing of Pinus pinaster or Eucalyptus globulus wood samples for enhancing their potential as hydrolysis substrates was considered previously 28-31 and two kinds of technologies were applied; alkaline treatments and delignification-swelling processes. Alkaline compounds (such a NaOH or Na2CO3) have been extensively studied as pretreatment agents for lignocellulosic materials. These kinds of compounds cause partial delignification and hemicellulose removal and modify the structure of lignocellulose, improving the behaviour of substrates towards the enzymic hydrolysis of cellulose. On the basis of previous studies, 28,29 the operational conditions listed in Table 1 were selected for performing the extraction of pine and eucalyptus wood with NaOH solutions. Figure 1 summarizes some simplified material balances describing the chemical alteration caused by these treatments on the solid phase. The chemical processing of wood in HOOCCH3H20-HCI media allows the separation of its main chemical fractions. Thus in a single treatment, the lignin is degraded to soluble fragments and hemicelluloses are solubilized, whereas cellulose remains in the solid phase. Since the

Table 1. Operational conditions used for the alkaline processing of pine and eucalyptus woods

NaOH concentration (%) Temperature (*C) Liquor:wood ratio, o.d. basis (g/g) Duration of treatment (h)

Pine

Eucalyptus

10 130 6 3

10 130 10 2

273

removal of lignin and hemicelluloses is extensive, the solid residue is notably enriched in cellulose, and fits the chemical requirements for good hydrolysis substrates. However, low yields were obtained in further saccharification experiments, owing to the unfavourable structural properties of delignified materials (essentially, high crystallinity and reduced surface available area). Both aspects can be improved by performing a subsequent swelling treatment with oxidizing or alkaline agents. 3°-32 The two-stage delignification-swelling processes for wood utilization are more complex than other alternatives presented in the literature, but present several advantages, including: (i) the lignin is recovered with little chemical alteration and results potentially useful for chemical purposes, (ii) the hemicellulose degradation products (sugars and/or furfural) have commercial value and can be easily separated from the lignin fraction by changing the polarity of the reaction medium, and (iii) the process is highly selective in relation to cellulose d e g r a d a t i o n . 26,33,34 Additionally, the main objective of the swelling treatments was to induce changes in the physicochemical properties of the substrates (and not to change their compositions, which were already favourable). The modification of structural factors such as crystallinity can be reached with low consumption of chemicals, allowing the utilization of recycle streams that limit the costs derived from this step. On the basis of previous studies,30, 31 NaC10 and NHaOH were selected as swelling agents for delignified pine and eucalyptus wood, respectively; and the operational conditions chosen for experimentation are listed in Table 2. Figure 2 shows some material balances describing the effects caused by these treatments.

Enzymic hydrolysis The solid residues obtained in the above treatments were used as hydrolysis substrates in media containing cellulase and cellobiase mixtures. The operational conditions for the saccharification step were chosen considering the dependence of both the polysaccharide conversion and the parameters describing the reaction kinetics on the operational vaI'iables. 28-31 Moreover, economic estimates 32,35 provided a useful guide for this purpose (especially for selecting the duration of treatments and the enzyme loading). Table 3 lists the operational conditions selected under which 43-66% polysaccharide conversion was obtained, leading to solutions with glucose concentrations in

J. C Parajret al.

274

NaOH solution

100 kg o. d. pine wood

>

ALKALINE EXTRACTION

42.9 kg cellulose 17.6 kg hemicelluloses 302 kg Ilgnln 9.3 kg other compounds

t

I

f

l

> Solid residue

t

35~1 kg cellulose 12.2 kg hemicelluloses 22.8 kg Ilgnln and other

TO ENZYMIC HYDROLYSIS

LJQUONb

Lignin degradation products Carbohydrate degradation products

NaOH solution

100 kg o. d. eucalyptus wood 43.8 kg cellulose 17.7 kg hemlcelluloses 26.0 kg Iignln 12.5 kg other compounds

>

ALKALINE EXTRACTION

l

I.iq~m t ~ractlv~ i.~nin ~radation

• Solid residue

33A kg cellulose 6.6 kg hemicelluloses 252. kg Ilgnln and other

TO ENZYMIC HYDROLYSIS

products Carbohydrate degradation products

Fig. 1. Simplified material balances describing the effects of the alkaline processing of pine and eucalyptus woods.

the range 18-43 g/litre. It should be noted that the glucose concentrations depended on several factors, including polysaccharide conversion, solid composition and liquor:solid ratio. An important aspect of the studied processes is that the sugar concentrations achieved in the enzymic hydrolysis are of the same order as those utilized in most studies reported on the fermentative production of lactic acid. Fermentation assays The enzymic hydrolysates obtained as described earlier were supplemented with nutrients, sterilized and used as carbon sources for lactic acid

Table 2. Operational conditions used for the delignificationswelling treatments of pine and eucalyptus woods

Delignification treatment Acetic acid concentration (%) Catalyst (HC1) concentration (%) Temperature (*C) Liquor:wood ratio, o.d. basis (g/g) Duration of treatment (h) Swellingtreatment Swelling agent Concentration (mol/litre) Temperature (°C) Liquor:wood ratio, o.d. basis (g/g) Duration of treatment (h) Initial pH

Pine

Eucalpytus

95 0.45 130 8 0"5

95 0.2 110 10 1

NaCIO 0-34 40 10 2 8

NH4OH 4 60 10 3 -

-

Lactic acidfrom wood NsCIO soluUons

Acetic acld:wster:.HCI solutions

100 kg

j

l

o. d. pinewood

> DELIGNIFICATION 42.9 kg cellulose 17.6 ko hemicelluloess 30.2 kg Ilgnln 9.3 kg other compounds

275

t

1

Solid residue 34~ kg cellulose 32. kg hemicelluloess 4.6 kg lignln and other

,J "L SWELLING Liquors

Solid residue 34.3 kg cellulose 0.7 kg hemlcelluloses 0.6 kg Ilgnln and other

Liquors J Extractlves Ugnln degradation products

TO ENZYMIC HYDROLYSIS

Carbohydrate

degradation products

NH4OH solutions

Acetic acld:waten.HCI solutions

100 kg o. d. eucalyptus wood

l

> DELIGNIFICATION

I l

t43.8 kg cellulose

17.7 kg hemk:elluloess 26.0 kg Iignin 12.5 kg other compounds

Solid residue 43.0 kg cellulose 4.1 kg hemicelluloess 3.0 kg Ilgnin and other

d"t SWELLINGt ~ Liquors

Solid residue 43.0 kg cellulose

3A kg hemicelluloess Liquors Extractives Llgnln degradation products Carbohydrate degradation products

Fig.2.

1.5 kg Ilgnin and other

TO ENZYMIC HYDROLYSIS

Simplified material balances describing the effects of the delignification-swellingtreatments of pine and eucalyptus

woods. production. In order to obtain comparative data, culture media were formulated from standard glucose solutions (20 or 50 g/litre), which were supplemented with the same type and concentration of nutrients than those used for media made from hydrolysates. From a qualitative viewpoint, the fermentation trials performed with the various culture media showed a similar kinetic pattem, with a close interrelationship between the concentrations of

biomass and product and an almost quantitative consumption of substrate after the exponential growth phase. A number of mathematical models have been proposed to describe the fermentative production of lactic acid. 6,7,1°-21 As a general strategy for analysis of data, the estimation of the kinetic parameters included in these models required the smoothing of experimental data by logistic equations and/or numerical integration of results. In

J. C Paraj6et al.

276

our case, the data were adequately modelled from the ideas of Mercier et aL6 following a simplified calculation scheme that avoids both the smoothing and numerical integration steps. The dependence of the biomass concentration (variable X) on the fermentation time was interpreted using the equation of Moraine and R o g o v i n , 36 that was previously employed for correlating data of lactic fermentations. 6,7 The corresponding expression is:

XmaxX)

dX-/~maxX( 1 - d t

,

(')

= PoP 1 Pmax ' Table 3. Operational conditions used for the enzymic

hydrolysis of processed wood

Delignified-swelled

pH Cellulase loading (EPU/g) Cellobiase loading (IU/g) Temperature (°C) Liquor :wood ratio (g/g) Duration of hydrolysis (h)

Pine

Eucalyptus

Pine

Eucalyptus

4-85 6

4"85 6

4-85 13

4"85 13

169

X-

80

80

48-5 20

48.5 20

48-5 12

48.5 12

48

72

48

48

XoXm exp(gmaxt) Xm - X0 + X0 exp(#maxt)

(3)

PoPmexp(P'0t) P=Pm- Po+ Po exp(P'0t)"

(1)

where/~max is the maximum specific growth rate and Xm.xis the maximum biomass concentration. Mercier et al. 6 proposed a similar expression for modelling the course with time of the lactic acid concentration (variable P):

NaOH-treated

where Pm,x is the maximum concentration and P~ is defined as the ratio between the initial volumetric rate of product formation (rp) and the initial product concentration P0. Both eqns (1) and (2) can be directly solved to give the following expressions:

(4)

From the series of experimental data biomass concentration/time and lactic acid concentration/ time, the model parameters X0, Xmax,/~max,P0, Pmax and P~ can be calculated for each fermentation media by nonlinear regression using the leastsquares method. Tables 4 and 5 list the numerical values of the regression parameters calculated for the various experiments (expressed as the mean of duplicate assays), as well as their respective 99% confidence intervals. The same Tables list the statistical values of R 2 (measuring the degree of correlation) and Fischer's 'F' parameter measuring the statistical significance of equations. The substrate utilization by Lactobacillus amylophilus may be interpreted by the equation: dS dt

dP dX Ys/P.--+dt Ys/x dt '

(5)

80

where S is the substrate concentration and Ys/P and Ys/x are the inverses of the product and biomass yields, respectively.6 Preliminary attempts on the modelling of our experimental data with these equations considering Ys/P and Ys/x as optimiza-

Table 4. Results obtained by regression analysis of data biomass concentration/time determined for the various fermentation

media (values calculated from duplicate experiments)

Fermentation medium a

3(0 (g/litre) Opt. value

SG50 SG20 ATPW ATEW DSPW DSEW

0.173 0.116 0-034 0-047 0.239 0.188

99% Conf. limits 0.111-0.233 0-034-0-198 -0.01-0-080 -0.04-0.136 0-131-0.346 0.076-0.301

X,,ax (g/litre)

P,,ax (h- I)

Opt. value

99% Conf. limits

Opt. value

99% Conf. limits

7.69 3-75 3-11 2.95 6.47 6.62

7.12-8.26 3.25-4.24 2.71-3.52 2.50-3-39 6.06-6.87 5.96-7.28

0-373 0.595 0.849 0-737 0.448 0.449

0.328-0.417 0.444-0.746 0-578-1.119 0.381-1.093 0.375-0.520 0.356-0.542

R2

F

0-998 0.989 0.986 0.984 0.996 0.994

3321 562 396 242 1561 1079

aNotation used for fermentation media: SG50 = standard glucose solutions, 50 g/litre; SG20 = standard glucose solutions, 20 g/litre; ATPW= alkali-treated pine wood; ATEW= alkali-treated eucalyptus wood; DSPW--delignified and swelled pine wood; DSEW= delignified and swelled eucalyptus wood.

Lactic acidfrom wood

277

table 5. Results obtained by regressionanalysisof data lactic acid concentration/timedetermined for the various fermentation media(valuescalculatedfrom duplicate experiments) Fermentation medium a

SG50 SG20 ~TPW ATEW DSPW DSEW

Po (g/litre)

Pmax (g/litre)

P'o (h- 9

Opt. value

99% Conf. limits

Opt. value

99% Conf. fimits

Opt. value

99% Conf. limits

0"368 0'626 0"201 0"173 0"794 0"506

0"218-0"519 0"206-1"045 0"039-0"364 0"006-0"338 0"499-1"089 0"187-0-826

46"54 23"01 14"59 15"83 44"28 41'22

43"74-49"33 17"94-28-08 12"95-16"23 14"41-17"26 41"98-46"59 37"35-45"09

0"443 0"501 0-747 0-693 0-456 0"488

0"399-0"488 0"369-0"633 0"583-0-911 0"531-0"855 0"406-0"506 0"403-0"572

R2

F

0-999 0"989 0"991 0"997 0"998 0-997

5533 581 712 1204 3970 1832

'Notation for fermentationmedia as in Table 4. • 50~-- ~

6. Results obtained by regression analysis of data substrate concentration/product concentration determined for the various fermentation media (values calculated from duplicate experiments) Table

Fermentation medium a

A (dimensionless) Opt. value

SG50 SG20 ATPW ATEW DSPW DSEW

1.058 1.056 1"097 1.180 0.961 1.032

99% Conf. limits

1.033-1.084 1"009-1.103 1.029-1.163 1"125-1.236 0-930-0.992 1"004-1-060

X(calc)

A" "&' " '&" - & . . • .

"'" 4

S (exp)

sO,,,.'O -,&

,.el

30

s'

"• .,f

k 20

0.998 0.991 0.986 0"995 0"995 0"997

•

.... S (calc)

•

40

R2

o P(exp) -- P(calc)

X(exp)

no

a"

.,

...~£3S

~

.~g, Eg-.-~__~-e-e-eo

a~-O--O ~O

I

5

"'-

no

Fermentation time

aNotation for fermentation media as in Table 4.

• -...•.... ""A ••

-'-" .~ 15-

Os .p

"• "A

• • .

tion parameters showed good correlation, but low absolute values for Ys/x, with comparatively wide 99% confidence intervals for this coefficient (being the zero included in its confidence ranges). As a result, a simplification of eqn (5) was utilized: considering the first term of the right side of eqn (5), it can be followed that the substrate consumed can be expressed as a linear function of the product generated:

So- S = A ( P - Po),

(6)

where A is a regression parameter. Table 6 lists the numerical values of A calculated for the various experiments, its 99% confidence intervals and the corresponding values of R 2. As can be seen from Figs 3-5, eqns (3), (4) and (6) with the parameters of Tables 4-6 provide a quantitative interpretation of the process, with excellent agreement between experimental and calculated data for all the substrates assayed. The most important factors when measuring the potential of hydrolysates as fermentation

;~

r

13

I0-

•)¢0

~•. 5-

r~,j:r

• - ~-"Ja"

0

.. "_° '"a° ' ° "

~._~_o~o---w-

2

I

4

•,.

• o,...-*'°':°'. -°''-~ "k.

o. .

I 6

Fermentation

Fig. 3.

I

8

.

" "1 "

10

I ]2

time

Experimental and calculated dependence of the

concentrations of substrate, product and biomass on the fermentation time corresponding to standard glucose solutions (resultsobtained from duplicate experiments).

media are the maximum biomass concentration, the maximum specific growth rate, the volumetric productivity of lactic acid and the product yield. The following paragraphs include some discussion on these variables. The variation ranges determined in this work for the maximum biomass concentration (3-7.7 g/litre) and for the maximum specific growth rate (0.373-0-848 h -1) are in good agreement with

278

Z C. Paraj6 et al.

i

• X (exp) ~ X (calc)

IS!'

a P (exp) - - P (calc)

- S (exp) .... S (calc)

• --

50

Alkali-treated pine wood

,,,. A...A. ~,

~ s

a"A'.

t~

6

8

I0

,,.0,"

°

X

"...,p/

i 3o~

o,,J :r

s

5"

,

",..

6

,

o"

20

"~' D/4~ "s

a'..

,,r~i"A ..

O....O,-.-" 4

~..

S #

b.

a~ IO

~...

~ ,d

"•"'.A

1~S 2

,,

,..o..O...O - ' ' ' ~ -

".

//'% s s~

.~o--~---. 0--'0--0 "-o

50/~ Delignified and swelled eucalyptus wood 4OF-

..D--n

"..""•

no-

.

Fermentation time

Alkali-treated eucalyptus wood

• "---•...&.. • ..%. .--. 1 5 ..•

•...

sO 'S

..n "~

Fermentation time

20

.~' f~"..

,

4

,jr"

-....

20IO --

2

~ . , . a "1~

J

,a

o

• S (exp) . . . . S (calc)

~"

s°

"i.•

P (exp) P (calc)

"'-•--..

"--" 3 ( -

/

~"

" --

Delignified and swelled pine wood

,..,, 4 0 k"A..A

Os.Sd ~ . " ' 0 q s

•"•. " " 10

X (exp) X (calc)

8

lO

12

Fermentation time

Fig. 4. Experimental and calculated dependence of the concentrations of substrate, product and biomass on the fermentation time corresponding to hydrolysates of alkalitreated pine and eucalyptus woods (results obtained from duplicate experiments).

data determined using standard glucose solutions. 37 The higher specific growth rates were obtained using fermentation media made from hydrolysates of alkali-treated wood, a fact that could be related to a comparatively reduced product inhibition, owing to low glucose concentrations. The maximum volumetric productivities of lactic acid (2.7-5.2 g/litre h depending on the fermentation medium considered) were obtained after 5.7-10.9 h of fermentation time, reaching values near the 6.5 g/litre h reported for the same microorganism. 13 On the other hand, the mean volumetric productivities of lactic acid determined in this work (1.33-2.74 g/litre h) are closely related to the corresponding values reported for lactic fermentations (2-5 g/litre h). 38'39 The faster fermentation kinetics were determined for media obtained from wood subjected to alkaline processing. The glucose concentrations (S) at the start of the fermentation trials were lower than those obtained in hydrolysates, owing to the dilution effect caused by the inocula. The increases in lactic acid concentration reached at the end of fermen-

_~0~.

- - ~ O ~* ~m~o~m'g -*'-e-, O~O~O ~ 2

4

6

8

o~O~O

--

.#

10

12

I

~' " ' • - - a - 14

Fermentation time

Fig. 5. Experimental and calculated dependence of the concentrations of substrate, product and biomass on the fermentation time corresponding to hydrolysates of delignified and swelled samples of pine and eucalyptus woods (results obtained from duplicate experiments).

tation were of the same order as the initial glucose concentrations, indicating product yields near the theoretical maximum (1 g lactic acid/g glucose for homolactic fermentation). From the variables involved in eqn (6), it can be proposed that the inverse of A provides an estimate of the product yield Ye/s, and that the term (1 - 1/A ) measures the fraction of substrate used for cell growth and/ or for cell maintenance. The values calculated for the parameter A (listed in Table 6) suggest products yields in the range 0-85-0.97 for five fermentation media, these results being in reasonable agreement with reported data. 13,2° The value of A estimated for hydrolysates obtained from delignified and swelled pine wood can be justified by experimental error and/or by the possibility that the microorganism can use a part of the nutrients as a carbon source. In fact, Mercier et al., using a similar fermentation media, obtained lactic acid concentrations of the same order as the initial glucose concentrations. Additionally, these pine wood hydrolysates contain other monosaccharides different from glucose that could be converted into lactic acid.

Lactic acid from wood

In conclusion, this work explores an effective way for performing the bioconversion of wood (a renewable, widely available and cheap raw material) into lactic acid (a chemical with remarkable added-value). Both alkaline and delignification-swelling treatments of wood led to solid residues susceptible to enzymic hydrolysis, allowing the production of glucose solutions useful as fermentation media. The bioconversion of hydrolysates was adequately interpreted by models reported in the literature. For this purpose, a simplified calculation scheme was developed in order to avoid both the smoothing and the numerical integration of experimental data. The general trends observed in the fermentation runs were similar for all the culture media studied, with limited lag periods, almost quantitative substrate conversion after the exponential growth phase and product yields near the theoretical maximum. The kinetic parameters demonstrated the suitability of hydrolysates for bioconversion to lactic acid. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y ' X u n t a de Galicia' (Proj. X U G A 3 8 3 0 2 B 9 3 ) . Special t h a n k s to M r s

Rocfo Rodrfguez for her excellent technical assistance. REFERENCES 1. Lipinsky, E. S. & Sinclair, R. G., ls lactic acid a commodity chemical? Chem. Eng. Prog., 82 (1986) 26-32. 2. Chahal, S. E, Lactic acid. In Ullman's Encyclopedia of Industrial Chemistry. VCH, Berlin, 1989. 3. San Martin, M., Pazos, C. & Coca, J., Reactive extraction of lactic acid with alamine 336 in the presence of salts and lactose. J. Chem. Tech. Biotechnok, 54 (1992) 1-6. 4. Keller, A. K. & Gerhardt, E, Continuous lactic acid fermentation of whey to produce a ruminant feed supplement high in crude protein. Biotechnol. Bioeng., 17 (1975) 997-1018. 5. Mulligan, C. N., Sail, B. E & Groleau, D., Continuous production of ammonium lactate by Streptococcus cremoris in a three stage reactor. Biotechnol. Bioeng., 38 (1991)1173-81. 6. Mercier, E, Yerushalmi, L., Rouleau, D. & Dochain, D., Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus. J. Chem. Tech. Biotechnol., 55 (1992) 111-21. 7. Norton, S., Lacroix, C. & Vuillemard, J. C., Kinetic study of continuous whey permeate fermentation by immobilized Lactobacillus helveticus for lactic acid production. Enzyme Microb. TechnoL, 16 (1994) 457-66.

279

8. Hang, Y. D., Direct fermentation of corn to L( + ) lactic acid by Rhizopus oryzae. BiotechnoL Lett., 11 (1989) 299-300. 9. Hamamci, H. & Ryu, D. D. Y., Production of L(+ ) lactic acid using immobilized Rhhopus oryzae: reactor performance based on kinetic model and simulation. Appl. Biochem. Biotechnol., 44 (1994) 125-33. 10. Luedeking, R. & Piret, E. L., A kinetic study of the lactic acid fermentation batch process at controlled pH. J. Microb. Techn. Eng., 1 (1959) 393-412. 11. Luedeking, R. & Piret, E. L., Transient and steady states in continuous fermentation. Theory and experiment. J. Biochem. Microbiol. Technol. Eng., 1 (1959) 431-59. 12. Friedman, M. R. & Gaden, E. L., Growth and acid production by Lactobacillus (L.) delbrueckii in a dialysis culture system. Biotechnol. Bioeng., 12 (1970) 961-74. 13. Hanson, T. P. & Tsao, G. T., Kinetic studies of the lactic acid fermentation in batch and continuous cultures. Biotechnol. Bioeng., 14 (1972)233-52. 14. Rogers, P. L., Bramall, L. & McDonald, I. J., Kinetic analysis of batch and continuous culture of Streptococcus cremoris HP. Can. J. Microbiol., 24 (1978) 372-80. 15. Jorgensen, M. H. & Nikolajsen, K., Mathematic model for lactic acid with Streptococcus cremoris from glucose. Appl. Microbiol, Biotechnol., 25 (1987) 313-6. 16. Yeh, P. L. H., Bajpai, R. J. & Iannotti, E. L., An improved kinetic model for lactic acid fermentation. J. Ferm. Bioeng., 71 (1991)75-7. 17. Amrane, A. & Prigent, Y., Mathematical model for lactic acid production from lactose in batch culture: model development and simulation. J. Chem. Tech. Biotechnol., 60 (1994) 241-6. 18. Tayeb, J., Bouillanne, C. & Desmazeaud, M. J., Computerized control of growth with temperature in a mixed culture of lactic acid bacteria. J. Ferment. TechnoL, 62 (1984) 461-70. 19. Leh, M. B. & Charles, M., Lactic acid production by batch fermentation of whey permeate: a mathematical model. J. Ind. Microbiol., 4 (1989) 65-70. 20. Samuel, W. A., Lee, Y. Y. & Anthony, W. B., Lactic acid fermentation of crude sorghum extract. BiotechnoL Bioeng., 22 (1980) 757-77. 21. Aborhey, S. & Williamson, D., Modeling of lactic acid production by Streptococcus cremoris hp. J. Gen. Appl. Microbiol., 23 (1977) 7-21. 22. Abe, S. & Takagi, M., Simultaneous saccharification and fermentation of cellulose to lactic acid. Biotechnol. Bioeng., 37 (1991) 93-6. 23. Takagi, M., Inhibition of cellulase by fermentation products. Biotechnol. Bioeng., 26 ( 1984) 1506- 7. 24. Chosson, J., Inhibition on cellulolytic activity by lactate in a Cellulomonas uda species. Biotechnol. Bioeng., 29 (1987) 767-9. 25. Browning, B. L., Methods" of Wood Chemistry. John Wiley & Sons, New York, 1967. 26. Vfizquez, D., Lage, M. A., Paraj6, J. C. & Vfizquez, G., Fractionation of Eucalyptus wood in acetic acid media. Biores. Technol., 40 (1992) 131-6. 27. Sharpe, M. E., Fryer, T. F. & Smith, D. C., Identification of the lactic acid bacteria. In Identification Methods for Microbiologist, ed. A. Partie, B. M. Gibbs & F. A. Skinner. Academic Press, New York, 1966. 28. Paraj6, J. C., Alonso, J. L., Lage, M. A. & Vfizquez, D., Empirical modeling of Eucalyptus wood processing. Bioproc. Eng., 8 (1992) 129-36. 29. Paraj6, J. C., Alonso, J. U & Vfizquez, D., Effect of selected operational variables on the susceptibility of

280

30. 31.

32. 33.

34.

J.C. Paraj6 et al. NaOH-pretreated pine wood to enzymatic hydrolysis: a mathematical approach. Wood Sci. Technol., 28 (1994) 297-307. Paraj6, J. C., Alonso, J. L. & Santos, V., Analysis of kinetic data in the enzymatic hydrolysis of delignified wood. J. Wood Chem. Technol. (submitted). Paraj6, J. C., Alonso, J. L. & Santos, V., Delignification and swelling of Eucalyptus wood ahead of enzymatic hydrolysis of the cellulosic fraction. Proc. Biochem., 30 (1995) 537-45. Paraj6, J. C., Alonso, J. L. & Santos, V., Enzymatic hydrolysis of wood: an engineering assessment. Bioproc. Eng., 12 (1995) 253-61. Paraj6, J. C., Alonso, J. L. & V~zquez, D., On the behaviour of lignin and hemicelluloses during the Acetosolv pulping of wood. Biores. Technol., 46 (1993) 233-40. Paraj6, J. C., Alonso, J. L., V~izquez, D. & Santos, V., Optimization of catalysed Acetosolv fractionation of

pine wood. Holzfors., 47 (1993) 188-96. 35. Zacchi, G., Skoog, K. & Hahn-Hiigerdal, B., Economic evaluation of enzymatic hydrolysis of phenol-pretreated wheat straw. Biotechnol. Bioeng., 30 (1988) 460-6. 36. Moraine, R. A. & Rogovin, P., Kinetics of polysaccharide B-1459 fermentation. Biotechnol. Bioeng., 8 (1966) 511-24. 36. Gon~alves, L. M. D., Xavier, A. M. R. B., Almeida, J. S. & Carrondo, M. J. T., Concomitant substrate and product inhibition kinetics in lactic acid production. Enzyme Microb. TechnoL, 13 (1991) 314-9. 37. Suskovic, J., Beluhan, S., Beluhan, D. & Kurtanjek, Z., Mathematical model and estimation of kinetic parameters for production of lactic acid by Lactobacillus delbrueckii. Chem. Biochem. Eng. Q., 6 (1992) 127-32. 38. Xavier, A. M. R. B., Gonqalves, L. M. D., Sobreiro, A. C. C. J. & Carrondo, M. J. T., Extraction coupled high cell density bioreactor for lactic acid production. Biochem. Eng. (Stuttgart) ( 1991 ) 250-3.