Production of lactic acid by lactic acid bacteria T and Lactobacillus amylovorus DSM 20531 T Lactobacillus coryniformis subsp.

torquens DSM 20004

Antonija Trontel, Nua Jelovac, Marina Pupovac, Anita Slavica, Sran Novak* Laboratory of Biochemical Engineering, Industrial Microbiology, Malting and Brewing Technology, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6/IV, HR-10000 Zagreb, Croatia
*Correspondence to: S. Novak, srnovak@pbf.hr
INTRODUCTION
A variety of simpler and complex carbohydrates present in renewable and waste materials may be utilized in the production of lactic acid. The use of pure cultures of lactic acid bacteria capable of fermenting mono- and disaccharides and directly converting polysaccharides to lactic acid without the need for saccharification is advantageous because of the cost and efficiency of the bioprocess. Lactic acid bacteria Lactobacillus amylovorus DSM 20531T and L. coryniformis subsp. torquens DSM 20004T were used in this work to produce lactic acid from monosaccharide (glucose), disaccharides (sucrose, maltose or cellobiose) and polysaccharides (starch or cellulose) as a sole carbon and energy sources in the MRS medium. Likewise, combinations of the two carbohydrates were used for the fermentative production of lactic acid in the medium. The usage of cellobiose was of the greatest interest because it could be derived from cellulose, giving a plus to the sustainability of the bioprocess. Segments of the physiological characterization of the two lactic acid bacteria were covered in this work.

RESULTS
Table 1. Selected physiological characreristics of L. amylovorus DSM 20531T and L. coryniformis subsp. torquens DSM 20004T.

L. amylovorus DSM 20531T MRS media MRS-glc MRS-mal MRS-cb MRS-suc MRS-starch gS0 (g L-1) gS (g L-1) 20 10 20 20 20 0 10,07 9,44 10,02 n.d. n.d. gX (g L-1) 4,98 2,87 6,03 3,92 pH18** 3,77 6,09 4,16 4,50 6,20 6,43 gla (g L-1) 20,55 1,67 10,64 10,53 1,67 n.d.

L. coryniformis subsp. torquens DSM 20004T gS (g L-1) 10,63 5,64 19,18 10,42 n.d. gX (g L-1) 2,81 3,02 2,01 3,69 pH18** 4,50 4,98 6,18 4,42 6,36 gla (g L-1) 10,15 4,50 1,29 10,02 0,64

20,12*
20,90* 4,02 3,97 13,25* 11,08*

19,98*
20,34* 3,16 2,99 12,11* 10,56*

MRS-cl
MRS-glc/mal MRS-glc/cb MRS-glc/cl MRS-cb/cl

20
10/10 10/10 10/10 10/10

n.d.
4,36/6,23 2,50/10,73 3,54/n.d. 10,08/n.d.

6,46
4,40 4,62 4,64 6,28

0,61
10,12 8,08 7,20 0,94

0/10,26
0/10,10 0/n.d. 0/n.d.

4,30
4,33 4,37 4,38

11,42
11,74 11,94 11,40

n.d, not determined; *consisted of bacterial biomass and remaining starch or cl; **value determined after 18 h of cultivation

MATERIALS AND METHODS

Microorganisms Lactobacillus amylovorus DSM 20531T and L. coryniformis subsp. torquens DSM 20004T (DSMZ GmbH, Braunsweig, Germany) were used in this work. The strains were maintaned in MRS medium at 4C and propagated overnight at 30C before experiments. Cultivations Fermentations were carried out in 10 mL (test tubes) or 200 mL (Erlenmeyer flasks on magnetic stirrer) of MRS media (pH 6,2 0,2) containing different carbohydrates or their combinations (Table 1.). Methods Concentration of carbohydrates and products of fermentation were determined by high-pressure liquid chromatography (HPLC) using Shimadzu chromatograph (Shimadzu Class-VP LC-10AVP, Japan) and Supelcogel C-610H column with Supelcogel H guard column. The elution was done isocratically (F = 0,5 mL min-1) with 0,1% H3PO4. Estimation of biomass concentration was based on determination of optical density (OD600) and biomass dry weight (gX). Methods used here were perfomed as described previously (Trontel et al, 2010).

0,08
cb

0,06
U (V)

0,04 0,02 0,00

10 12 14

la aa

MRS 0h 2h 4h 6h 8h 10 h 12 h 14 h 24 h 26 h

4 3

14 12

10 8
6

2 1 0

4 2 0 0 5 10 15 t (h) 20 25 30

16 18 t (min)

20

22

24

Fig. 1. Part of chromatograms of the samples withdrawn during cultivation of L. amylovorus DSM 20531Tin MRS-cb medium.

Fig. 2. Changes in concentration of: substrate (gcb, ), biomass (gX, ), lactic acid (gla, ) and acetic acid (gaa, ) during cultivation of L. amylovorus DSM 20531T (full symbols) and L. coryniformis s ubsp. torquens D SM 20004T (open symbols) in the MRS-cb medium at 40C.

Table 2. Duration of growth phases and estimated values of some biokinetic parameters of growth and lactic acid production by L. amylovorus DSM 20531T and L. coryniformis subsp. torquens DSM 20004T.

growth of the bacteria microorganism tgrowth phase

lag exp

lactic acid production YX/S PrX (g L-1 h-1) 0,20 0,25 rP (h-1) 0,20 0,25

max
(h-1)

gXmax
(g L-1) 3,52 1,63

rS (h-1) 0,0800 0,0004

gPmax
(g L-1) 9,11 0,99

YP/S

YP/X

(h)
Abbreviations cb cellobiose cl cellulose glc glucose mal maltose suc sucrose Symbols PrX productivity of biomass (g L-1 h-1) PrP productivity of lactic acid (g L-1 h-1) rS substrate consumption rate (h-1) rP product formation rate (h-1) t time (h) YX/S biomass yield coefficient (g g-1) YP/X product/biomass yield coefficient (g g-1) YP/S product yield coefficient (g g-1) g concentration (g L-1 ) Subscripts aa acetic acid la lactic acid max maximum S substrate X biomass dry mass 0 initial

(g g-1) 0,23 0,08

(g g-1) (g g-1) (g L-1 h-1) 0,67 0,08 2,93 0,96 0,36 0,25

L. amylovorus DSM 20531T L. coryniformis subsp. torquens DSM 20004T

lag, lag phase; exp, exponential phase.

0 2

16 4

0,12 0,23

CONCLUSIONS
1. L. amylovorus DSM 20531T and L. coryniformis subsp. torquens 20004T can transport and homofermentatively metabolize selected mono- and disaccharides, sole carbon and energy sources added in MRS medium, to lactic acid. When present in the medium together with another substrate, glucose is preferable carbon source, except in MRS-glc/mal when both carbohydrates were simultaneously fermented by L. coryniformis subsp. torquens 20004T to lactic acid. In chosen conditions (test tube) the two bacterial strains can not utilise starch and cellulose. 2. Cellobiose (gcb0 = 12 g L-1) was completely depleted during 24 h of heterofermentation by L. amylovorus DSM 20531T and was stoichiometrically converted to lactic acid and acetic acid. Poor growth and fermentation of cellobiose by L. coryniformis subsp. torquens 20004T has to be further studied.

References Trontel, A., Bari, V., Slavica, A., antek, B., Novak S. (2010) Modelling the effect of different dubstrates and temperature on the growth and lactic acid production by Lactobacillus amylovorus DSM 20531T in batch process. Food Technol. Biotechnol. 48(3), 308-316.

gcb, gla, gaa (g L-1)

PrP

gX (g L-1)