Ethanol production from mahula (Madhuca latifolia L.) flowers using free and immobilized (in Luffa cylindrical L. sponge discs) cells of Zymomonas mobilis MTCC 92

Abstract

A novel immobilization method involving Zymomonas mobilis immobilized in a luffa (Luffa cylindrica L.) matrix for ethanol production from mahula (Madhuca latifolia L.) flowers was investigated. The dried spongy fruits of luffa, a cucurbitaceous crop available in abundance in tropical and sub-tropical countries, have been found to be a promising material for immobilizing microbial cells. In our study, the cells not only survived, but they were also active physiologically for four growth cycles, yielding ethanol at 251.1 ± 0.012, 247.9 ± 0.08, 243.4 ± 0.042 and 240.71 ± 0.033 g/kg flowers in the first, second, third, and fourth cycles, respectively, after 96 h fermentation. Ethanol production by the immobilized cells was 9.2% higher than that by free cells. The ethanol yield (Yp/s), volumetric substrate uptake (Qs), and final sugar to ethanol conversion rate (%) obtained with luffa-immobilized cells of Z. mobilis were 0.439 ± 0.004 g/g, 0.994 ± 0.009 g/l/h, and 87.8%, respectively, which were 7.06, 2.62, and 6.92% higher than that of free cells.

Introduction

In recent years, interest has been focused with renewed enthusiasm on the field of biomass and bioenergy, driven mainly by the recognition that the global crude oil reserve is finite and its depletion is occurring much faster than previously predicted. An additional factor is that environmental deterioration, resulting from the overconsumption of petroleum-derived products, especially the transportation fuels, is threatening the sustainability of human society (Bai et al. 2008). Ethanol, both renewable and environmentally friendly, is believed to be one of the best alternatives to petroleum products.

In this context, mahula (Madhuca latifolia L.) flowers (corolla), a rich source of fermentable sugars (glucose, fructose, and maltose) is a useful bio-resource (US$ 35–40 tonne^-1) for ethanol production through fermentation (Mohanty et al. 2009; Behera et al. 2010a). Mahula is a deciduous tree commonly found in the tropical rain forests of Asia and Australia. Bio-ethanol can be produced using either free or immobilized cells (Carvalho et al. 2002). Procedures for ethanol production involving the immobilization of whole cells provide several advantages: (1) the cell mass can be easily separated from the bulk liquid for possible reuse; (2) the system can function continuously over a prolonged period; (3) reactor productivity is enhanced; (4) catalytic efficiency is higher (Behera et al. 2010b). Different immobilization techniques, such as entrapment in Ca-alginate beads, agar–agar, k-carrageenan, among others, have been used extensively in fermentation industries for producing various bio-products, such as amino acids (Bodalo et al. 1996), enzymes (Kar et al. 2009), organic acids (John et al. 2007), and bio-ethanol (Behera et al. 2010a).

Luffa (Luffa cylindrica L., Family Cucurbitaceae) is a common Cucurbitaceous vegetable crop grown abundantly in tropical and sub-tropical countries (Ogbonna et al. 1997). It is a climbing plant that bears elongated and cylindrical berry-like fruits. The fibrous vascular network of the dried fruits of luffa can be used as a bath sponge and for the manufacture of table, door, and bath mats. The spongy nature of the dried fruits make them a suitable carrier matrix for use in microbial cell immobilization procedures (Ogbonna et al. 1994). Luffa sponge has high degree of porosity, a high specific pore volume, and stable physical properties, and it is biodegradable, non-toxic, and relatively inexpensive (Liu et al. 1998).

The yeast Saccharomyces cerevisiae has traditionally been considered to be the major ethanol-fermenting microorganism (Davis et al. 2005). In recent years, the Gram-negative anaerobic bacterium Zymomonas mobilis has also been applied in procedures involving ethanol fermentation (Behera et al. 2010b). This microorganism converts sugar almost stoichiometrically to ethanol and CO₂, growing more rapidly and demonstrating a higher ethanol productivity than S. cerevisiae (Queresi and Manderson 1995; Rogers et al. 1997; Davis et al. 2005; Davis et al. 2006). The ethanol tolerance of Zymomonas spp. is also equally as good as that of S. cerevisiae (Busche et al. 1992; Davis et al. 2006), and it produces fewer by-products (Nowak and Roszyk 1997). However, its catabolizing capabilities are restricted to a few substrates, such as glucose, fructose, maltose, and sucrose (Lee and Huang 2000; Ruanglek et al. 2006). Ethanol production by free and immobilized cells of Z. mobilis has been studied using several substrates, i.e., molasses (Panesar et al. 2001), paper sludge (Yamashita et al. 2008), cassava chips and flour (Nellaiah and Gunasekaran 1992), and mahula flowers (Behera et al. 2010b). In the latter study, Z. mobilis MTCC 92 entrapped in Ca-alginate matrix was studied for its potential for ethanol production and the results compared with that of free cells (Behera et al. 2010b).

The objective of the experiments reported here was to study ethanol production from mahula flowers using both free cells of Z. mobilis MTCC 92 and those immobolized in a luffa sponge disc, in a submerged fermentation system. The growth and fermentation kinetics between the free and immobilized bacterial cells were compared.

Materials and methods

Mahula flowers

Fresh mahula flowers were collected from the forests of Keonjhar district of Orissa, India, during March–April, 2009. The flowers were brought to the Microbiology Laboratory of Department of Botany, Utkal University, Bhubaneswar, washed in tap water to remove dust and other debris and sun-dried in the open air for 7 days to reduce the moisture content to 11%. The sun-dried flowers collected from various locations were mixed thoroughly before being used for ethanol fermentation. The composition of the sun-dried flowers [expressed in g/100 g dry weight (DW) basis] was: moisture, 11.0 ± 0.32; starch, 0.9 ± 0.007; total sugar (glucose, fructose, sucrose, and maltose), 61 ± 0.34; crude protein, 6.5 ± 0.27; crude fiber, 15.3 ± 0.5; total ash, 1.8 ± 0.31; undetermined solids, 3.5 ± 0.42. Total sugar, starch, and crude protein were estimated by the methods of Mahadevan and Sridhar (1999), and the remaining parameters were estimated by the methods described by Amerine and Ough (1984). The pH of the mahula flower was measured in a slurry of flowers and water in 1:5 ratio.

Microorganism and culture condition

Zymomonas mobilis MTCC 92, procured from the Institute of Microbial Technology, Chandigarh, India, was maintained on yeast extract glucose salt agar (YGSA) medium [(g/l): yeast extract, 10; glucose, 20; MgCl₂, 10; (NH₄)₂SO₄, 10; KH₂PO₄, 10; agar, 15] with the pH adjusted to 6.5. The culture was stored at 4 ± 0.5°C for further use.

Preparation of inoculum

The inoculum prepared in 250-ml Erlenmeyer flasks containing 100 ml of liquid growth medium (as mentioned above but without agar) that had been sterilized at 121°C for 20 min. Each flask was inoculated with a loopful of the Z. mobilis culture and incubated for 24 h at 30°C at 120 rpm in an orbital shaker incubator (Remi Pvt, Ltd, Bombay, India). The resulting culture served as the inoculum for the free cells used in the ethanol production experiments.

For immobilized cells, 60 ml of bacterial inoculum [equivalent to 10% of the fermentation medium (600 ml)] was immobilized with the luffa discs as matrix as described in the following section.

Immobilization of whole cells in luffa sponge discs

The luffa sponge discs were obtained from mature dried fruits of L. cylindrica. The sponge was cut into discs, each with a diameter of 2.5 cm and a thickness of 4 mm, and washed four time in boiling water. The luffa discs were then oven-dried at 70°C. A 60-ml sample of the bacterial inoculum was directly poured into six luffa sponge discs contained in a 500-ml beaker. After the cells (inoculum) had become trapped within the matrix of the sponge, the immobilized luffa discs were removed from the beaker and washed thoroughly with fresh sterilized YGSA broth to remove any remaining free cells before being used in the fermentation experiments. The flow-chart for the Z. mobilis cell immobilization method in luffa sponge discs is given in Fig.1.

Fig. 1

Process of immobilization of Zymomonas mobilis cells in luffa sponge discs

Fermentation medium

Mahula flowers (100 g) were ground (flower:water ratio, 1: 5, w/v) in a mixer–grinder (TTK Prestige, Bangalore, India) to obtain an uniform slurry. (NH₄)₂SO₄ was added to the slurry at the rate of 1 g/l as a source of nitrogen for Z. mobilis, and the pH was then adjusted to 6.5 with the addition of 1 N NaOH. The slurry (600 ml) was poured into 1-l Erlenmeyer flasks and inoculated with 10% or 60 ml of bacterial inoculum in the case of the free cell fermentation experiments and with six immobilized luffa sponge discs in the case of the immobilized cell fermentation experiments. This procedure (600 ml medium in 1-l Erlenmeyer flask) was identical to that used in previous experiments (Behera et al. 2010a, b). An optimal culture environment was achieved based on the <90% conversion rate of sugar to ethanol by Z. mobilis (Behera et al. 2010b). Each experiment was run in triplicate (n = 3 flasks) for the free and immobilized cell experiments, respectively, for 96 h at room temperature (30 ± 2°C).

Analytical methods

At 24 h post-culture initiation, the fermented broths (in triplicate flasks) were removed and the contents analyzed for total sugar and ethanol. The ethanol content of the fermented broth was determined by measuring the specific gravity of the distillate according to the procedure described by Amerine and Ough (1984). In this procedure, the weight of a certain volume of an alcohol distillate is compared to the weight of precisely the same volume of distilled water. The ratio of the weights of the two (alcohol:water) give the specific gravity of the distillate. The total sugar content was assayed by anthrone method (Mahadevan and Sridhar 1999). The pH was measured using a pH meter (Systronics, Ahmadabad, India) fitted with a glass electrode. The luffa sponge was dried at 80°C for 24 h prior to the immobilization procedure, and the initial weight was determined. After immobilization, the luffa sponge containing the immobilized the cells was washed gently with distilled water and dried at 80°C for 24 h. The immobilized cell concentration was calculated from the difference in the weight of the luffa sponge before and after immobilization. The immobilized cells, separated after the fermentation procedure, were reused for successive three batches. The fermentation kinetics were studied as per the formulae given by Bailey and Ollis (1986).

Statistical analysis

The data of ethanol production using free and immobilized cells of Z. mobilis were analyzed using one-way analysis of variance (ANOVA). Where a significant difference in ANOVA (p < 0.05) was detected by the Fisher’s least significance difference (LSD), a multiple comparison test was applied to compare the factor level difference. The analysis was performed using MSTAT-C (ver. 2.0; Michigan State University, East Lansing, MI).

Results and discussion

The main fermentable sugar components of the mahula flowers were glucose and fructose (Swain et al. 2007). In order to compare the fermentation efficiency, the mahula flowers were fermented with free and immobilized cells of Z. mobilis MTCC 92 and the sugar utilization and ethanol production by free and immobilized cells compared (Fig. 2).

Fig. 2

Study of ethanol production by free (a) and luffa-immobilized cells (b) of Z. mobilis by mahula flowers

Both free and luffa-immobilized cells of Z. mobilis MTCC 92 started growing in the log phase, achieving maximum ethanol production during the stationary phase (96 h) (Fig. 2). The initial sugar concentration was 61 ± 0.34 g/100 g mahula flowers, which is significantly higher than the 36–38 g/100 g mahula flowers observed in our previous studies (Swain et al. 2007; Behera et al. 2010a). This difference may be due to variations in mahula plant genotype and/or collection from different localities. There was a fall of 53.6 and 55.74% in total sugar concentration relative to the initial content with a concomitant production of 97 ± 0.016 and 114.75 ± 0.062 g ethanol kg⁻¹ mahula flowers up to 24 h of fermentation by the free and immobilized bacterial cells, respectively. A decrease in the sugar reserve may also be due to its utilization by the microorganisms for growth and metabolism in addition to its conversion into ethanol (Behera et al. 2010b). After 24 h, there was a gradual increase in ethanol concentration with increasing incubation period accompanied by a simultaneous decrease in total sugar contents (Fig. 2). After 96 h of fermentation, 81.72 and 87.8% of the initial sugar content had been converted, resulting in 228 ± 0.008 and 251.1 ± 0.012 g ethanol/kg mahula flowers in the systems using free and luffa-immobilized cells of Z. mobilis, respectively [45 kg of fermentable sugar (as glucose) yields 20-30 kg of ethanol; Reed 2002]. Thus, ethanol production was 9.2% higher in the luffa-based immobilization system than in the free cell system; this difference was statistically significant (Fischer’s LSD test, p < 0.05, LSD between treatments 3.79). Swain et al. (2007) reported that the average yield of ethanol from fresh and 12-month-stored mahula flowers using immobilized (in Ca-alginate matrix) yeast cells was 206 and 152 g ethanol/kg flowers, respectively; this was 6.3 (193 g ethanol/kg flowers) and 2.63% (148 g ethanol/kg flowers) more than that produced by fresh and 12-month-stored free cells. Behera et al. (2010a) reported that ethanol production by yeast cells immobilized in agar–agar (151.2 g ethanol/kg flowers) and Ca-alginate (154.5 g ethanol/kg flowers) was 1.39 and 3.5% higher than that by free cells (149.1 g ethanol/kg flowers) after 96 h of fermentation. On the other hand, Ogbonna et al. (2001) showed that luffa sponge was an excellent cell carrier for ethanol fermentation by flocculating (S. cerevisiae) and non-flocculating (Candida brassicae) cells. Ogbonna et al. (1997, 2001) further confirmed that luffa sponge alone can be used to achieve 99% immobilization of flocculating yeasts (S. cerevisiae) cells for ethanol production in a column bioreactor.

The immobilized cells were recycled three more times (total n = 4), limiting the duration of each fermentation cycle to 96 h as most of the sugars in mahula flowers were consumed during that period. The cells not only survived but were also active physiologically, producing 247.9 ± 0.08, 243.4 ± 0.042 and 240.71 ± 0.033 g ethanol/kg flowers in the second, third, and fourth cycle relative to the first cycle (251.1 ± 0.012 g ethanol/kg flowers) after 96 h of fermentation; this was a respective decrease in ethanol production of 1.28, 3.06, and 4.14%. This decrease may be due to the marginal leakage of cells from the luffa matrix during each batch of fermentation. In terms of ethanol production, similar results were obtained from cane molasses using an alginate–luffa carrier matrix for the immobilization of yeast cells (Phisalaphong et al. 2007). In this latter study, the ethanol production was the same during the first and second cycle of operation (91.7 g ethanol/l cane molasses), with a marginal decrease (0.5%) in the third cycle (90.6 g ethanol/l cane molasses).

The growth and fermentation kinetics of free and immobilized cells were also studied (Table 1). The ethanol concentration (P) obtained with luffa-immobilized cells of Z. mobilis (41.85 ± 0.013 g/l) was 9.2% more than that obtained using free cells (38 ± 0.012 g/l), whereas the volumetric substrate uptake (Qs) was found to be 2.62% higher with immobilized cells (0.994 ± 0.009 g/l/h) than with free cells (0.968 ± 0.008 g/l/h). Likewise, the ethanol yield (Yp/s = 0.439 ± 0.004 g/g) and volumetric product productivity (Qp = 0.436 ± 0.008 g/l/h) obtained with luffa immobilized cells were 7.06 and 9.2% higher than those of free cells (Yp/s =0.408 ± 0.01 g/g; Qp = 0.396 ± 0.011 g/l/h). Likewise, the final sugar to ethanol conversion rate (%) with immobilized cells was 6.92% higher than that with free cells of Z. mobilis. However, the final biomass (X) concentration in the case of immobilized cells (5.42 ± 0.04 g/l) was considerably lower than that of free cells (6.37 ± 0.024 g/l), which was useful during product separation and the purification process (Diderich et al. 1999).

Table 1 Growth and fermentation kinetics of free and immobilized cells of Zymomonas mobilis MTCC 92 on mahula flowers

Mahula trees have been an intrinic part of the sustainability of the tribal people for centuries in Asia and Australia (Swain et al. 2007), but its commercial potential has not been fully explored. The mahula flower has great potential as a substrate in the industrial-scale production of ethanol given the ever-increasing demand for ethanol and the enormous availability of this cheap raw material on the Indian subcontinent (Mohanty et al. 2009). Thus, the cost of savings from the use of cheap raw materials, such as mahula, must come hand in hand with a cheap and efficient fermentation process. A variety of different factors, such as high surface area per volume, strong and durable structure, low-specific gravity (which makes it light), and reasonable cost, are the characteristics of luffa which make it a suitable alternative for use as an immobilization matrix for ethanol fermentation (Oboh and Aluyor 2009). In our study, luffa-immobilized bacterial cells produced 9.2% more ethanol than free cells, signifying that luffa sponge was an excellent support and a promising method for Z. mobilis immobilization in procedures aimed at producing ethanol from mahula flowers. The utility of luffa sponge as an immobilizing matrix has also been studied for other fermentative products. Slokoska and Angelova (1998) reported that luffa sponge could be used as an ideal immobilization material for pectinase production. Vignoli et al. (2006) reported that sorbitol production from sucrose was twofold higher (50 g/l) using luffa-immobilized cells of Z. mobilis than in the free cells (25 g/l). Likewise, luffa sponge-immobilized fungal biosorbents have been used extensively for the biosorption of heavy metals from olive oil mill waste water and other waste waters (Iqbal and Edyvean 2004; Iqbal and Edyvean 2005; Ahmadi et al. 2006a, b). Luffa sponge fibers have traditionally been used for bathing and dishwashing; more recently, the fibers have been used for environmental reclamation (Iqbal and Edyvean 2004).

Conclusion

A novel immobilization method of Z. mobilis in a luffa matrix for ethanol production from mahula flowers was investigated. The results demonstrate that luffa sponge is a very effective immobilization carrier. Since dry luffa is tasteless, odorless, and colorless and pre-treatment with chemicals is not required, this material has a great potential for use in the fermented food and beverage industries. The method described here can also be easily scaled up for industrial ethanol fermentation processes in tropical and subtropical countries, where luffa grows in abundance. In comparison with the commercially available synthetic immobilization carriers, the use of luffa sponge will not lead to any additional technical problems during scale-up process. Furthermore, the low cost and the ease with which immobilization can be achieved means that adaptation of this process in developing tropical countries is very feasible.