LACTIC ACID FERMENTATIVE PRODUCTION USING WASTE FROM THE HARVEST OF GREEN SUGAR CANE AS A SUBSTRATE

Liliana Serna Cock and Aida Rodríguez de Stouvenel

Liliana Serna Cock. Bacteriologist, Universidad Católica de Colombia, Bogotá, Colombia. Doctor in Food Engineering, Universidad del Valle (Univalle), Cali, Colombia Professor. Engineering Faculty, Universidad Nacional de Colombia, Campus of Palmira, Colombia, carrera 32 Vía Candelaria, Palmira, Colombia. e-mail: lsernac@palmira.unal.edu.co

Aida Rodríguez de Stouvenel. Chemical Engineer, Univalle, Cali, Colombia. Doctor in Science, Université Catholique de Louvain. Professor Department of Food Engineering, Univalle, Cali, Colombia. aidrodri@univalle.edu.co

SUMMARY

Waste products from the harvest of green sugar cane (var. CC 85-92), were tested as a substrate in batch type fermentative production of lactic acid. The fermentations were carried out at 32°C, pH 6.0, with juice obtained from cane tops and leaves (JTL) and Lactococcus lactis subs lactis isolated from the same variety of cane. Lactic acid concentration (LA), substrate conversion (SC), biomass concentration, velocity of product formation (r_p) and yield (Y_p/s) were measured, and the results were compared with those of fermentations using the same strain in MRS culture medium, with 60g·l^-1 of glucose. Lactic acid concentrations up to 28.5g·l^-1 with a yield of 0.85g·g^-1 were obtained using JTL medium in a 48h incubation period. JTL and MRS showed statistically significant differences in Y_p/s, SC and biomass concentration, while JTL and MRS were not significantly different in LA and r_p, suggesting that waste products from the sugar cane harvest could be used as a cheap raw material for the fermentative production of lactic acid.

RESIDUOS DE CAÑA DE AZÚCAR COMO SUSTRATO EN LA PRODUCCIÓN FERMENTATIVA DE ÁCIDO LÁCTICO

RESUMEN

Para la producción fermentativa de ácido láctico se evaluaron como sustrato residuos de cosecha de caña de azúcar (variedad CC85-92). Las fermentaciones se realizaron a 32°C y pH 6,0, utilizando jugos obtenidos de hojas y cogollos (JTL), y Lactococcus lactis subs. lactis aislado de caña de azúcar de la misma variedad. En las fermentaciones se midieron la concentración de ácido láctico (LA), la conversión de sustrato (SC), la concentración de biomasa, la velocidad de formación de producto (rp) y el rendimiento (Yp/s), y los resultaron se compararon con fermentaciones que utilizaron la misma cepa y medio de cultivo comercial MRS adicionado de glucosa hasta 60g·l^-1. Utilizando JTL se pueden obtener concentraciones de ácido láctico por encima de 28,5g·l^-1 y rendimientos de 0,85g·g^-1 en 48 horas de fermentación. Los sustratos JTL y MRS mostraron diferencias estadísticamente significativas en Yp/s, SC y concentración de biomasa, y diferencias estadísticamente no significativas en LA y rp. Los resultados sugieren que los residuos de cosecha de caña de azúcar pueden ser utilizados como materia prima barata para la producción fermentativa de ácido láctico.

RESÍDUOS DE COLHEITA DE CANA DE AÇÚCAR COMO SUBSTRATO na PRODUÇÃO FERMENTATIVA DE ÁCIDO LÁCTICO

RESUMO

Para a produção fermentativa de ácido láctico se avaliaram como substrato, resíduos de colheita de cana de açúcar (variedade CC85-92). As fermentações se realizaram a 32°C e pH 6,0, utilizando sucos obtidos de folhas e brotes (JTL), e Lactococcus lactis subs. lactis isolado de cana de açúcar da mesma variedade. Nas fermentações se mediram a concentração de ácido láctico (LA), a conversão de substrato (SC), a concentração de biomassa, a velocidade de formação de produto (rp) e o rendimento (Yp/s), e os resultados se compararam com fermentações que utilizaram a mesma cepa e meio de cultivo comercial MRS adicionado de glicose até 60g·l^-1. Utilizando JTL se podem obter concentrações de ácido láctico por encima de 28,5g·l^-1 e rendimentos de 0,85g·g^-1 em 48 horas de fermentação. Os substratos JTL e MRS mostraram diferenças estatisticamente significativas em Yp/s, SC e concentração de biomassa, e diferenças estatisticamente não significativas em LA e rp. Os resultados sugerem que os resíduos de colheita de cana de açúcar podem ser utilizados como matéria prima barata para a produção fermentativa de ácido láctico.

KEYWORDS / Green Sugar Cane / Lactococcus lactis / Sugar Cane Leaves / Sugar Cane Tops /

Received: 03/03/2006. Modified: 02/16/2007. Accepted: 03/19/2007

Introduction

The practice of burning the sugar cane fields during harvest has been widespread in the Colombian sugar agro-industry since the early 1970s (Villegas, 2002). This harvesting practice increases atmospheric CO₂, causes accidental forest fires and generates social disapproval. However, it has been accepted because the burning of the cane before it is cut eliminates between 15 and 20ton/ha of total waste products, as the fire mainly consumes the dry leaves. Thus, the volume of waste products is lower than when the cane is harvested green. Colombian environmental laws (MMA, 1997) prohibits even controlled burning, and for this reason the sugar cane agro-industry is in a state of transition towards the establishment of a production system using green cane. Sugar mill technicians agree that the greatest obstacles to the implementation of a production system using green cane are the management of harvest waste products, the pulling out of the cane roots and the ground preparation in fields that are going to be replanted (Villegas, 2002). The quantity of plant residues left in the field after harvesting green cane depends on variety, soil fertility and whether the harvesting process is manual or mechanized (Victoria et al., 2002); Among the varieties studied in Colombia, V7151 generates the most residues (65ton/ha), while the MZC74275 variety produces 32.2ton/ha of green material. Part of the waste is made up of sugar cane left in the fields. In the case of CC85-92, the most commonly cultivated variety in Colombia, 4.5ton/ha of cane stems are left in the field when harvested by machine, while manual harvesting leaves 3.5ton/ha of the product. (Victoria et al., 2002).

The wastes produced during the harvesting of the cane are chopped up and then spread over the field to be planted, or in the paths between the furrows. This process, when harvesting green cane, requires an investment 3-5 times greater than in the case of burnt cane. For the majority of mills, the additional cost per ha is between USD 26.32 and 35.1 (Villegas, 2002). For this reason, research is underway to find means of accelerating the decomposition of green waste.

The waste material from green harvesting has a water content of ~75% and a nutritional content of total sugars, nitrogen, phosphorous, potassium, calcium and magnesium. These nutrients are necessary for microbe growth, which suggests that the waste products from the cane harvest could be used as cheap substrates for fermentation (Villegas and Torres, 1999).

One of the problems with fermentative production of lactic acid is the high cost of the substrate. Akerberg and Zacchi (2000) showed that the highest operational cost is that of the raw material, while Kwon et al. (2000) indicated that the yeast extract alone represents more than 30% of production costs.

The potential of the waste products from sugar cane harvest as a substrate for the batch type fermentative production of lactic acid, was evaluated in the present study.

Materials and Methods

Lactococcus culture

Lactococcus lactis subs lactis was selected from 20 lactic acid bacteria (LAB) isolated from sugar cane of the CC85-92 variety (Serna-Cock and Rodríguez de Stouvenel, 2006).

Fermentation

The two substrates used for fermentation were juice extracted from tops and leaves (JTL) and commercial MRS culture medium, recommended for the growth of lactic acid bacteria (De Man et al., 1960). The tops and leaves were waste products from the harvesting of green sugar cane of the CC85-92 variety, provided by the Centro de Investigación de la Caña de Azúcar (CENICAÑA), Candelaria, Valle del Cauca, Colombia.

The JTL was obtained by pressing three loads with an experimental press, and then sterilizing the juice for 10min at 121°C. The MRS culture medium was prepared according to commercial guidelines (Merck, 1994), sterilized as above, and pure glucose added to 60g·l^-1.

According to Serna-Cock and Rodríguez de Stouvenel, (2006) the optimal conditions (32°C, pH 6.0 and a glucose concentration of 60-65g·l^-1) for lactic acid production for this strain were used. The pH was adjusted with NaOH 5M, in 500ml conical flasks, with a working volume of 250ml. For both substrates, 10% of inoculla were used with a fermentation time of 72h and an agitation velocity of 120rpm.

Analytical method

The concentrations of total sugars (glucose, fructose and sucrose) and lactic acid were measured using high performance liquid chromatography (HPLC; Hitachi L-6000A, integrator D-2500, Tokyo, Japan; equipped with a column for acid and sugar, Aminex HPX 87H, 300mm), and using sulfuric acid 0.005M as the mobile phase.

The biomass was calculated from optical density data at 540nm (DO₅₄₀) using a spectrophotometer (Milton Roy 401, Rochester, USA). In order to establish a linear correlation between optical density and biomass values, optical density and driveway of the cells were measured at 0, 2, 4, 6, 9, 12, 24, 48 and 72h of fermentation. All measurements were made in triplicate. The estimated correlation was utilized to convert all the DO₅₄₀ values to biomass concentration. However, at the end of each one of the fermentations, broth samples were taken and the lactic biomass and optical density determined to corroborate the results.

The percentage of substrate conversion (SC) and yield (Y_p/s) in g·g^-1 were calculated using the expressions

where S₀: initial total sugar concentration (g·l^-1), S: final total sugar concentration (g·l^-1) at time when P is maximum, P: lactic acid concentration (g·l^-1). Velocity of product formation, r_p, was derived from the equations for the kinetic data obtained (g·l^-1·h^-1).

Statistical design

The data on LA, SC, biomass concentration, r_p and Y_p/s were analyzed using single factor variance analysis with two levels: juice extracted from tops and leaves (JTL), and commercial culture medium (MRS)

Results and Discussion

The kinetics of product formation, substrate consumption and biomass production for JTL and MRS are presented in Figure 1, and the calculated kinetic parameters in Table I. In both substrates, lactic acid production was associated with growth of Lactococcus lactis.

Variance analysis showed that the substrate has a highly significant effect on SC, biomass concentration and Y_p/s (P<0.005) and less marked effect occurs for LA and r_p.

The lactic acid (LA) concentrations reported in this study are analogous with those obtained by Yoo et al. (1997), who evaluated the efficiency of various sources of nitrogen for obtaining LA from glucose; they obtained concentrations of 34.5 g·l^-1 with corn steep liquor, 24.7g·l^-1 with soy peptone, 33.7g·l^-1 with Primatone 6.7, 22.1g·l^-1 with casamine acid and 32.3g·l^-1 with N-Z amine. The results for LA concentrations obtained in the present study were superior to those obtained by Tik et al. (2001), who reported 25.59g·l^-1 of LA in extractive fermentations, with sunflower oil added as an immobilizing agent.

When the LA concentrations obtained from green sugar cane harvest residues were compared with the results in studies using other substrates, it was found that LA concentrations obtained after 36h of fermentation, and using 10g·l^-1 of starch, wheat, yucca (manioc) and potato (10.05, 7.82, 7.85, 4.72 and 4.42g·l^-1, respectively; Xiaodong et al., 1997) were far lower than those obtained using JTL. Oda et al. (1997), reported much lower concentrations and yields (0.13g·l^-1 and 47.2%) using corn steep liquor added to bread crust; after 72h of fermentation.

Table II shows LA concentrations, product yields and substrate conversions obtained by other authors using agro-industrial by-products and waste products.

LA concentrations obtained with MRS are comparable to those reported by Kurbanoglu and Kurbanoglu (2003) of 36g·l^-1 in commercial medium (CM), but these authors obtained greater LA concentrations using fibrous protein (ram horns) as a nitrogen source.

The product yields obtained agree with those reported by Gonçalves et al. (1997) of 0.56 and 0.75g·g^-1, using 130g·l^-1 of pure glucose in fermentations at pH 5.0 and 6.0, respectively. Yumato and Ikeda (1995), cited in Vishnu et al. (2000), reported yields of 0.6g·g^-1 with 50g·l^-1 of soluble starch and 0.68g·g^-1 with 45g·l^-1 of maize starch, using an amylolytic strain.

Bearing in mind that no nutritional supplement was added to JTL, and that this material is a waste product from the harvest normally left in the field, the fact that there were statistical differences compared to the MRS substrate suggests that the juice extracted from cane tops and leaves could provide a cheap raw material for the commercial production of lactic acid. This finding ratifies the idea put forward by Sreenath et al. (2001) who used alfalfa harvest residues to produce lactic acid and concluded that these could be used as a substrate for fermentation in large-scale lactic acid production.

The results presented here also suggest the importance of orienting research toward the enrichment of juices made from tops and leaves with other sources of nitrogen, which would permit the fermentation to continue until the carbon source is completely consumed, since there is an undesirably high content of residual sugars (25.7g·l^-1) present in the separation process. Research is also needed on the behaviour of this substrate in continuous fermentations, on the recovery and separation operations, and also on the behaviour of other highly productive lactic acid bacteria that are commercially available. The harvest residues are also suited for the production of lactic biomass, as the yields obtained with MRS were far lower than those obtained using JTL.

Acknowledgements

The authors thank the Centro de Investigación de la Caña de Azúcar, (CENICAÑA) and Ingenio La Cabaña for their help in carrying out this study.

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