Biotechnology Letters 25: 19831987, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.

1983

Rhizopus arrhizus a producer for simultaneous saccharication and fermentation of starch waste materials to L(+)-lactic acid
Bo Jin1, , Li Ping Huang1,2 & Paul Lant1
1 Department of 2 School of

Chemical Engineering, The University of Queensland, St Lucia, QLD 4072, Australia Environmental Science and Engineering, Dalian University of Technology, Dalian, 116023, P.R. China Author for correspondence (Fax: +61-7-3365 4199; E-mail: bojin@cheque.uq.edu.au)
Received 1 July 2003; Revisions requested 23 July 2003/29 August 2003; Revisions received 22 August 2003/30 September 2003; Accepted 2 October 2003

Key words: L(+)-lactic acid, Rhizopus arrhizus, simultaneous saccharication and fermentation, starch waste efuents Abstract Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharication and fermentation process using starch waste efuents. Lactic acid at 19.544.3 g l1 with a yield of 0.850.96 g g1 was produced in 40 h using 2060 g starch l1 . Supplementation of nitrogen source may be unnecessary if potato or corn starch waste efuent was used as a production medium.

Introduction Lactic acid (2-hydroxypropionic acid) has a wide range of applications in food, pharmaceutical and cosmetics industries (Oda et al. 2002, Hofvendahl & Hahn-Hagerdal 2000). It has gained prominence in research and industry as a potential source for biodegradable and biocompatible poly-lactate polymers, which is an environmentally friendly alternative to plastics derived from petrochemical materials (Datta et al. 1995). Lactic acid occurs naturally in two optical isomers, D()- and L(+). L-Lactic acid is the preferred isomer in the food and pharmaceutical industries (Akerberg et al. 1998, Hofvendahl & Hahn-Hagerdal 2000). L -Lactic acid is commercially produced using lactic acid bacteria or fungi in submerged cultures. Among the Rhizopus fungi, R. oryzae has received the greatest interest and strain NRRL 395 has been recognised as one of the most suitable lactic acid producers (Oda et al. 2002, Tsai & Moon 1998, Yin et al. 1997, Yu & Hang 1989, Zhou et al. 1999). The production of lactic acid by Rhizopus fungi offers several benets over bacterial production processes. Bacterial fermentations have higher production costs due to the

pretreatment of raw materials and the supplementation of complex nitrogen components, such as yeast extract (Akerberg et al. 1998, Yin et al. 1997, Zhou et al. 1999). Furthermore, fungal cultures, such as Rhizopus oryzae, are amylolytic and they can produce lactic acid from various starchy materials, such as potato starch, without prior saccharication (Tsai & Moon 1998, Yu & Hang 1989). The objective of this study was to determine the feasibility of producing L-lactic acid by a newly identied fungus strain of Rhizopus arrhizus using waste starch materials. The ability of the fungal lactic acid producer using starch and nitrogen sources was investigated in shake-ask cultures.

Materials and methods Microorganism and production medium Rhizopus arrhizus DAR 36017, from Orange Agricultural Institute, Sydney, Australia, was grown on three production media: potato starch efuent, corn starch efuent and soluble starch medium. The potato and corn starch efuents were collected from waste streams of industrial potato and corn processes in

1984 Brisbane, Australia. The characteristics of the starch efuent were reported previously (Huang et al. 2003). The soluble starch medium consisted of (g l1 ) soluble starch 20, yeast extract 5, peptone 5, KH2 PO4 0.3, MgSO4 7H2 O 0.2 and ZnSO4 7H2 O 0.04. Cultivation method The soluble starch medium was inoculated with about 105 spores ml1 , incubated with shaking (150 rpm) at 30 C for 16 h and 5 ml then inoculated into a 250 ml ask containing 100 ml production media. The production medium had 1% (w/v) CaCO3 to maintain the pH around 5.5. Cultures were shaken at 150 rpm and at 30 C. Sample preparation Samples of cultures were ltered (100 ml) through a 106 m sieve. Biomass concentration was determined by weighing the mass after washing with 4 M HCl and distilled water to remove the residual calcium carbonate, and then drying at 60 C for 36 h. Part of the ltered liquid was centrifuged at 2350 g for 10 min and the supernatant was further ltered through a 0.22 m lter and then diluted ve times for lactic acid analysis. Analytical methods Starch was analysed using the iodine colorimetric method and reducing sugars were estimated by the dinitrosalicylic acid method using glucose as standard. Lactic acid, fumaric acid, formic acid, succinic acid and ethanol were measured by HPLC. The optical isomer of the lactic acid produced by Rhizopus arrhizus 30617 was tested by HPLC using the CHIRALPAK MA (+) column as detailed by Yin et al. (1997). The lactic acid produced by R. arrhizus 30617 was judged to be 100% Lisomer.

Fig. 1. Kinetic prole of simultaneous saccharication and fermentation of starch to L(+)-lactic acid by R. arrhizus 36017 using potato starch efuent containing 20.5 g starch l1 .

producing 0.2 kg biomass m3 h within 48 h. Lactic acid was produced at 1.1 kg m3 h over 12 h28 h. From 28 h to 48 h lactic acid increased from 18.2 to 19.8 g l1 with a yield rising from 0.89 to 0.97 g g1 starch. Effect of starch sources and concentration The concentration of lactic acid and fungal biomass increased from 8.2 to 44.2 g l1 and from 1.2 to 6.9 g l1 , respectively, as the initial starch concentration was increased from 10 to 60 g l1 (Figure 2). The highest lactic acid yield of approx. 0.96 g g1 starch was obtained with starch initially at 20 g l1 for both potato and corn starch efuent media. A slightly higher production rate of lactic acid and fungal biomass was obtained in the corn starch medium (Figures 2b and 2c). The highest yield of lactic acid was obtained as the starch concentration varied from 20 to 40 g l1 in the soluble starch medium presumably because this starch is more accessible to the enzymes secreted during the fungal growth.

Results Kinetic characteristics of simultaneous saccharication and fermentation As shown in Figure 1, R. arrhizus 36017 readily saccharies potato starch to glucose, which is then converted into lactic acid. Hydrolysis was approx. 1.5 kg starch m3 h over 12 h, resulting in reducing sugar accumulating at 4 g l1 . The cells grew rapidly

1985
Table 1. Effect of supplementation of (a) (NH4 )2 SO4 and (b) urea on production of lactic acid and other products by R. arrhizus 36017 using soluble starch medium containing 20 g starch l1 in 42 h fermentation. (a) Addition of (NH4 )2 SO4 (g l1 ) 0.5 1 2 3 5 (b) Addition of urea (g l1 ) 0.5 1 2 3 5 Concentration of products (g l1 ) Fungal Lactic Fumaric Formic biomass acid acid acid 0.7 1.3 1.5 2 2.2 6.3 8.2 10.9 14.7 16.2 0.4 0.5 0.7 1.4 2.4 0.4 0.9 1.7 1.9 Concentration of products (g l1 ) Fungal Lactic Fumaric Formic biomass acid acid acid 0.8 1.5 1.9 2.3 2.7 12.9 18.3 19.8 18.6 15.8 0.7 1.5 2.3 3.2 4.1 0.8 1.9 2.2 2.2

Succinic acid 0.3 0.5 0.7 0.7 0.8

Ethanol

1.3 1.3 1.3 1.3 1.4

Succinic acid 0.5 0.5 0.6 0.7 0.6

Ethanol

0.8 1.1 1.3 1.4 1.4

Table 2. Effect of supplementation of (a) yeast extract and (b) peptone on production of lactic acid and other products by R. arrhizus 36017 using soluble starch medium containing 20 g starch l1 in 42 h fermentation. (a) Addition of yeast extract (g l1 ) 1 3 5 8 10 (b) Addition of peptone (g l1 ) 1 3 5 8 10 Concentration of products (g l1 ) Fungal Lactic Fumaric Formic biomass acid acid acid 0.8 1.5 1.9 2.7 3.8 4.4 10.1 17.8 18.8 18.9 0.6 1.2 1.7 2.7 3.8 0.2 0.6 1.2 1.7 Concentration of products (g l1 ) Fungal Lactic Fumaric Formic biomass acid acid acid 0.9 1.7 2.2 3 3.3 4.6 12.2 18.9 19.1 19.2 0.9 1.4 1.8 2.9 3.6 0.1 0.5 0.7 1

Succinic acid 0.4 0.6 0.7 0.8 0.8

Ethanol

0.8 1.2 1.8 2.1 2.7

Succinic acid 0.3 0.5 0.5 0.7 0.7

Ethanol

0.7 0.9 1.1 1.9 2.2

1986 there was only a minor impact on ethanol production. The lactic acid concentration did not signicantly vary as the yeast extract and peptone concentrations varied from 5 to 10 g l1 (Table 2). An increase from 2 to 5 g (NH4 )2 SO4 l1 decreased the lactic acid concentration. Compared with (NH4 )2 SO4 and urea, yeast extract and peptone stimulated the fungal cell growth, resulting in a high concentration of fungal biomass produced. As a major by-product, fumaric acid was produced at a relatively high concentration in a medium containing high amounts of nitrogen source. Ethanol and other organic acids were produced at very low levels.

Discussion R. arrhizus 36017 has a high enzymatic and metabolic capability to produce lactic acid using pure or waste starch materials, corresponding to a starch conversion rate of 8595% in 40 h. Lactic acid yield of 0.95 g g1 starch was achieved using soluble starch medium with the addition of 2 g ammonium sulphate l1 or 5 g of either yeast extract or peptone l1 . However, supplementation by a nitrogen source may be unnecessary if starch waste efuent was used for lactic acid production. The strain R. arrhizus 36017 may be a specic producer of lactic acid with very low levels of other organic acids and ethanol being produced. Two signicant advantages of using this strain are that (1) it has a high enzymatic and metabolic capacity to carry out a single process for simultaneous saccharication and fermentation of starch materials to lactic acid, and (2) it can utilize raw and/or waste starch materials as a substrate, and the addition of low concentrations of simple organic and inorganic nitrogen sources can enhance the lactic acid yield. This may be a promising fermentation process for commercial lactic acid production. The volumetric concentration of lactic acid achieved was 2045 g l1 with increasing starch concentrations from 20 to 60 g l1 in 40 h fermentation. Lactic acid yield and starch conversion efciency decreased with increasing concentrations of all starch substrates. This may be due to a low accessibility of amylolytic enzymes to substrates and product inhibition during the later stages of fermentation at high substrate concentration (Petruccioli et al. 1996). Rhizopus arrhizus is well known for its capacity to produce fumaric acid (Martak et al. 2003, Petruccioli et al. 1996, Soccol et al. 1994). With the addition of CaCO3 , R. arrhizus MUCL 16179, R. arrhizus

Fig. 2. Effect of carbon sources (a) soluble starch, (b) potato starch and (c) corn starch, and starch concentration on lactic acid yield (Ya) and concentration (Ca), and fungal biomass concentration (Cb) by R. arrhizus 36017 in 42 h fermentation.

Effect of nitrogen source and concentration R. arrhizus 36017 utilized (NH4 )2 SO4 , urea, yeast extract and peptone for lactic acid production in the soluble starch medium, containing (g l1 ) soluble starch 20, KH2 PO4 0.3, MgSO4 7H2 O 0.2 and ZnSO4 7H2 O 0.04 (Tables 1 and 2). The urea was the least favoured nitrogen source. The concentration of lactic acid, fungal biomass and other by-products was increased as these nitrogen sources increased but

1987 MUCL 28425 and R. arrhizus NRRL 1526 metabolizes glucose or corn starch to produce a large amount of fumaric acid (Soccol et al. 1994, Yin et al. 1997). Riscaldati et al. (2000) reported that ammonium fumarate was produced by Rhizopus arrhizus NRRL 1526 under phosphate-limited conditions. Only a few R. arrhizus strains can produce lactic acid using glucose and corn starch while still producing fumaric acid (Rosenberg & Kristokova 1995). Lactic acid was produced by R. arrhizus CCM 8109 in a stirred tank reactor and the process was carried out under a periodical bleed and feed condition associated with an operation strategy of specic pH control and nitrogen source supplementation (Martak et al. 2003).
Hofvendahl K, Hahn-Hagerdal B (2000) Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb. Tech. 26: 87107. Huang L, Jin B, Lant P, Zhou J (2003) Biotechnological production of lactic acid integrated with potato wastewater treatment by Rhizopus arrhizus. J. Chem. Technol. Biotechnol. 78: 899906. Martak J, Schloaaer S, Sabolova E, Kristokova L, Rosenberg M (2003) Fermentation of lactic acid with Rhizopus arrhizus in a stirred tank reactor with a periodical bleed and feed operation. Proc. Biochem. 26: 111. Oda Y, Saito K, Yamauchi H, Mori M (2002) Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae. Curr. Microbiol. 45: 14. Petruccioli M, Angiani E, Federici F (1996) Semi-continuous fumaric acid production by Rhizopus arrhizus immobilized in polyurethane sponge. Proc. Biochem. 31: 463469. Riscaldati E, Moresi F, Federici F, Petruccioli M (2000) Direct ammonium fumarate production by Rhizopus arrhizus under phosphorous limitation. Biotechnol. Lett. 22: 10431047. Rosenberg M, Kristokova L (1995) Physiological restriction of the L-lactic acid production by Rhizopus arrhizus. Acta Biotechnol. 15: 367374. Soccol C, Stonoga V, Raimbault M (1994) Production of L-lactic acid by Rhizopus species. World J. Microbiol. Biotechnol. 10: 433435. Tsai S, Moon S (1998) An integrated bioconversion process for production of L-lactic acid from starchy potato feed stocks. Appl. Biochem. Biotechnol. 7072: 417428. Yin P, Nishina N, Kosakai Y, Yahiro K, Park Y, Okabe M (1997) Enhanced production of L(+)-lactic acid from corn starch in a culture of Rhizopus oryzae using an air-lift bioreactor. J. Ferment. Bioeng. 84: 249253. Yu R, Hang Y (1989) Kinetics of direct fermentation of agricultural commodities to L(+)-lactic acid by Rhizopus oryzae. Biotechnol. Lett. 11: 597600. Zhou Y, Dominguez J, Cao N, Du J, Tsao G (1999) Optimization of L-lactic acid production from glucose by Rhizopus oryzae ATCC 52311. Appl. Biochem. Biotechnol. 77-79: 401407.

Acknowledgements This study was funded by an Early Career Research Grant, The University of Queensland, Australia. We would also like to thank Freer Foods Pty Ltd. and Golden Circle, Brisbane, Australia for the supply of starch stream samples.

References
Akerberg C, Hofvendahl K, Zacchi G, Hahn-Hagerdal B (1998) Modelling the inuence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat our. Appl. Microbiol. Biotechnol. 49: 682690. Datta R, Tsai SP, Bonsignor P, Moon S, Frank J (1995) Technological and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol. Rev. 16: 221231.