Appl Microbiol Biotechnol (1990) 33:31-35

Applied Microbiology Biotechnology

Springer-Veriag 1990

Sugar-cane molasses fermentation by Zymomonas mobilis

Monica B. Doe|le and Horst W. Docile
Department of Microbiology, University of Queensland, St. Lucia-Brisbane, Queensland 4067, Australia Received 10 October 1989/Accepted 8 December 1989

Summary. Two different quality types of sugar-cane molasses containing a total sugar content of 48%-50% (w/v) and 35%-42% (w/v) were investigated for Zymomonas bioethanol production. Molasses concentrations of up to 250 g/1 (1:3 dilution) were successfully fermented within 24 h despite a higher salt concentration in the lower grade molasses. Higher molasses concentrations (300 g/l) led to fructose accumulation. The addition of sucrose to a final sugar concentration of 15% (w/v) led to 10% (v/v) ethanol with conversion efficiencies up to 96%. Sorbitol levels were negligible, but increased up to tenfold u p o n addition of invertase.

Introduction
Molasses of any type is undoubtedly the cheapest feedstock for ethanol fermentation (Maiorella et al. 1981; Kosaric et al. 1981), and is a by-product or waste product of the sugar-cane industry (Paturau 1982). The total sugar content of the various types of molasses varies between 40%-50% (w/v) with an ash content between 6%-15%. Molasses has been used for decades to produce rum ( M u r p h y 1984) or ethanol (Potgeiter 1981). Higher d e m a n d s for ethanol, however, increased the search for better fermentation conditions. Realizing that ionic salts are required but also function as inhibitors depending on their concentrations (Jones and Greenfield 1984), pre-treatment ( K a p o o r and Misra 1980; Rhee et al. 1984; Saigal and Viswanathan 1982), fed-batch (Hsie and Su 1980; Koshimizu et al. 1984; Borzani 1987), continuous fermentations (Murdiyatmo and Tedjowalijono 1987; Rogers 1985; Grote and Rogers 1985), use of higher yeast concentrations (Queiroz et al. 1983), cell recycle (Sedha et al. 1984; Wang et al. 1984), yeast selection (Sharma et al. 1980; H a r a l d s o n and Bjorling 1981; Converti et al. 1985) and mixed yeast (Shvets et al. 1985; Jones et al.
Offprint requests to: H. W. Doelle

1982; Haraldsen and Rosen 1982; Layokun 1984) or yeast and Zymomonas (Cruz and Borzani 1980) cultures were investigated together with the addition of invertase (Park and Sato 1982; Vitolo et al. 1985) and the effect of f o a m formation and suppression (Schugerl 1985) to find ways of improving the fermentation. Attempts with Z. mobilis as the fermenting microorganism led to incomplete sucrose hydrolysis (Gunasekaran et al. 1986; M u r p h y 1988a-c), low conversion efficiencies despite adaptation and nutrient supplementation (van Vuuren and Meyer 1982), and required expensive desalting pre-treatment (Rhee et al. 1984). This paper describes the first fully successful molasses fermentation by the Z. mobilis strain reported to be successful in sugar-cane syrup bioethanol production (Doelle and Doelle 1989).

Materials and methods

Microorganism. The organism used throughout these investigations was Z. mobilis UQM 2716, a laboratory strain developed from Z. mobilis UQM 2007 (NCIB 11199) and now deposited as ATCC 39676 (US patent no. 4797360). Maintenance. Zymomonas mobilis was maintained both in a liquid culture medium and on agar plates stored at 4 C. The liquid culture contained 100 g/1 sucrose, 10 g/1 glucose, and 2 g/1 each of MgSOa-7H20, KH2PO4, (NH4)2SO4, casein hydrolysate, and yeast extract. The culture was kept at 35 C and transferred at regular time intervals during the experiments. The agar plates contained 40 g/1 glucose as carbon source plus the same maintenance medium as the liquid culture with t7 g/1 Bacto agar. The cultures were transferred to another plate every 3-4 weeks. Bioprocess. Initial experi~aents were carried out using 500-ml flasks with the initial pH manually adjusted to 6.5 with 4.0 M NaOH after inoculation: incubation of the standing cultures was at 35 C. Batch cultivations were carried out in 3-1 Chemap (M/innedorf, Switzerland) bioreactors with automatic pH and temperature control. Without the addition of air or nitrogen, a stirrer speed of 90-100 rpm maintained homogeneous nutrient distribution, and the bioprocess temperature was maintained at 35C. After inoculation the pH was adjusted to 6.5 with no further con-

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trol. The feedstock molasses used was of two different types, classified in Australia as B- and C-molasses. Whereas B-molasses contains on average a total sugar concentration of 48%-50% ( w / v), which is equivalent to a value of 67%-70% (w/w), C-molasses possesses a much reduced sugar concentration of approximately 35%-42% (w/v), which is equivalent to 55%-60% (w/w). The Cmolasses could be regarded as closest to "blackstrap molasses". The molasses was diluted to the appropriate sugar concentration and used without heat treatment, sterilization or nutrient addition. The same procedure was adopted upon additional sucrose addition to bring the total sucrose content to 15% (w/v). Inoculation was carried out using an 8-h-old Z. mobilis culture grown on the above maintenance medium.

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Fig. 2. Fermentation pattern of Z. mobilis ATCC 39676 on 250 g/1 C-molasses: (3, sucrose; O, glucose; A, fructose; A, sorbitol; [], ethanol Ethanol ( f i n a l ) - ethanol (initial) = C Conversion efficiency (%)= C 100/A+ B

Results

Analytical methods. Sucrose, glucose, fructose, sorbitol and ethanol were analysed according to Doelle and Greenfield (1985) using HPLC and gas chromatography. Ethanol was additionally analysed by an ebulliometer, and initial sucrose was estimated by the invertase method (Lyness and Doelle 1981). Conversion efficiency was calculated as follows: Sucrose (g/l) 0.538 = A [glucose+fructose (g/l)] = B

A series of different-type molasses concentrations was chosen between 100-300 g/l, varying in total sugar concentrations between 40 g/1 ( = 100 g/l C-molasses) to 144 g/1 ( = 300 g/l B-molasses). The highest sugar concentration of C-molasses used was 123 g/1 ( = 3 0 0 g/1 C-molasses). The concentrations were chosen on the understanding that present molasses fermentations with yeast are conducted with t:4 and 1:5 dilutions of the original substrate. Figures 1 and 2 exhibit typical fermentation graphs obtained with 250 g/l (w/v) B- and C-molasses. The highest ethanol production rate with B-molasses was 0.59%/h or 4.66 g/1 per hour, which dropped to 0.32%/ h or 2.5 g/1 per hour, whereas with C-molasses the

Table 1. Fermentation patterns of Zymomonas mobilis ATCC 39676 using various concentrations of C-molasses (C-mol), C-molasses plus invertase, C-molasses plus sucrose ( = 150 g/l total sugar) and C-molasses plus sucrose in the presence o f invertase Serial no. C-Mol added (g/l) Sucrose added (g/l) Sucrose Initial (g/l) 42.09 78.99 93.10 110.77 45.55 78.99 93.10 110.77 143.64 142.50 t31.48 157.70 165.68 164.16 147.44 158.84 Final (g/l) 2.09 3.05 3.61 0.17 1.64 3.23 3.53 0.13 3.45 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Glucose Initial (g/l) 4.04 8.08 10.10 12.12 4.04 8.08 10.10 12.12 --------Final (g/l) 1.10 1.00 1.38 2.94 0.00 0.00 0.00 1.16 4.14 5.72 9.97 11.62 0.00 3.17 5.66 9.07 Fructose Initial (g/l) 4.04 8.08 10.10 12.12 4.04 8.08 10.10 12.12 --------Final (g/l) 0.00 0.15 5.93 26.49 0.05 0.19 11.96 40.81 0.00 0.57 8.54 29.75 0.1t 7.87 28.46 50.31 Final sorb (g/l) Final EtOH (g/l) Ferm time (h) Conversion efficiency (%)

CM1000 CM1020 CM1040 CM1060 CM1010" CM1030 a CM1050 a CM1070 a CM600 CM610 CM620 CM630 CM650" CM660 a CM670" CM680 ~

100.00 200.00 250.00 300.00 100.00 200.00 250.00 300.00 100.00 200.00 250.00 300.00 100.00 200.00 250.00 300.00

------~ -103.80 66.00 47.50 29.00 103.80 66.00 47.50 29.00

1.65 1.64 1.99 4.25 3.46 5.66 6.12 8.08 15.58 10.29 10.68 12.15 21.18 18.84 17.89 18.86

26.77 49.77 53.I7 48.98 28.63 49.51 52.54 51.82 68.73 74.26 73.47 63.99 79.00 80.42 67.15 54.51

15.50 24.00 24.00 40.00 15.50 24.00 24.00 40.00 24.00 24.00 40.00 40.00 24.00 40.00 40.00 40.00

99.90 98.00 88.00 68.09 99.00 97.50 92.45 73.38 84.56 92.68 98.99 7L42 85.37 87.84 81.08 60.54

" 0.125 g/1 invertase added Sorb, sorbitol; EtOH, ethanol; ferm = fermentation

33 Table 2. Fermentation patterns of Z. mobilis ATCC 39676 using various concentrations of B-molasses (B-tool) in the presence or absence of sucrose and invertase Serial no. B-Mol added (g/t) Sucrose added (g/l) Sucrose Initial (g/l) 46.77 93.54 116.93 140.31 146.88 142.63 Final (g/l) t.22 0.00 0.00 0.67 0.35 1.32 Glucose Initial (g/l) 0.25 0.50 0.63 0.75 3.41 5.44 Final (g/l) 1.48 0.80 2.65 3.52 3.96 0.73 Fructose Initial (g/l) 2.98 5.96 7.45 8.94 10.89 12.48 Final (g/l) 0.00 0.25 0.90 5.24 t.93 6.21 Final sorb (g/l) 0.00 4.78 4.98 6.29 7.79 16.39 Final EtOH (g/l) 35.55 56.88 67.55 74.66 79.00 82.56 Ferm time (h) 22.00 16.00 23.00 23.00 20.00 20.00 Conversion efficiency (%) 88.89 88.96 86.78 81.41 84.11 90.34

CM1230 CM1500 CM1510 CM1520 CM1650 CM1660a

100.00 -200.00 -250.00 -300.00 -2 5 0 . 0 0 30.00 2 5 0 . 0 0 30.00

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highest production rate occurred between 6 and 10 h with 0.5%/h or 3.95 g/l per hour, which dropped thereafter to 0.24%/h or 1.93 g/1 per hour. The results in Table 1 for C-molasses exhibit a very familiar trend, whereby the conversion efficiency drops from almost 100% to 68.1% with increasing molasses concentration. The sharpest decline occurred between 250 g/l and 300 g/1 molasses with the former giving higher ethanol yields. The main feature o f this fermentation was the extremely low sorbitol levels, which slightly increased to 0.4% with 300 g/l molasses. A further important observation is related to the actual fermentation time. Whereas 100-250g/1 molasses fermented out within 24 h, with 300 g/l molasses 40 h were required to obtain the low conversion efficiency o f 68.1%. The slow sucrose hydrolysis was enhanced by the addition o f 0.125 g/1 invertase. Although a slight increase in conversion efficiency and ethanol yield occurred (Table 1), the trend stayed the same and fermentation time did not improve. The addition of invertase did, however, double the sorbitol formation. Each molasses concentration was then supplemented with sucrose to a final concentration o f 150 g/1 total sucrose in order to test the possibility of a 10% (v/v) ethanol yield [ = 79-80 g/1 (w/v)]. This meant the addition o f 103.8, 66.0, 47.5 and 29.0 g/1 sucrose, respectively. The results in Table 1 indicate that the etha-

nol yield could be reached with invertase a d d i t i o n to 100 and 200 g/l molasses, but not with higher concentrations. In comparing fermentation with and without sucrose addition, the conversion efficiencies o f the lower molasses concentrations dropped, whereas the higher molasses concentrations increased significantly, in particular the 250 g/1 C-molasses. The drop in conversion efficiency was undoubtedly due to the increased sorbitol levels, which varied between 1% (without invertase) and 2% (with invertase addition). Although these experiments showed that 10.2% (v/ v) ethanol can be obtained within 24-40 h using 100200 g/l (w/v) C-molasses plus sucrose with an approximately 96% conversion efficiency, they also revealed that it is fructose uptake that causes problems in molasses fermentation. 'With sucrose completely hydrolysed with all molasses concentrations, the residual fructose levels are the reason for retardation o f the efficiency in higher molasses concentrations. B-Molasses is a higher quality molasses with a total sucrose content o f approximately 50% (w/v). The fermentation pattern of 250 g/1 B-molasses (Fig. 1) shows the same trend as for C-molasses alone. The fermentation results are given ~n Table 2. In using the same molasses concentrations (100-300 g/l), the conversion efficiencies are very much closer and within 10% compared to 30% in the case of C-molasses. In contrast to the Cmolasses pattern, fermentation was complete within
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B-molasses plus 30 g/l sucrose: O, sucrose; O, glucose; A, fructose; i , sorbitol; [3, ethanol

Fig. 4. Fermentation pattern of Z. mobilis ATCC 39676 on 250 g / l B-molasses plus 3 0 g / l sucrose and 0.125 g/1 invertase: O, sucrose; O, glucose; A, fructose; i , sorbitol; El, ethanol

34 24 h with conversion efficiencies ranging between 80% and 90% depending u p o n the molasses concentration. Figures 3 and 4 demonstrate the B-molasses fermentation pattern using B-molasses plus sucrose to a total sugar content of 150 g/1 with and without invertase addition. The highest ethanol production rates were very similar with 0.94%/h [ = 7.4 g/1 per h o u r ( w / v)] and 0.97%/h [ = 7.6 g/1 per hour (w/v)] respectively. At higher B-molasses concentrations problems were also observed in regard to fructose uptake with sorbitol levels slightly higher, although to a less p r o n o u n c e d extent than with C-molasses. conversion efficiency and thus final ethanol yields significantly (Borrego et al. 1988), but m a y be able to reduce residence time (Amin et al. 1987; Amin and Doelle 1989), although these systems have not been tried on feedstocks such as molasses. The lower cell biomass requirement of Z. mobilis in comparison to yeast (Wang et al, 1984) certainly saves the industry recycling equipment capital costs. We were unable to confirm earlier reports of extensive filamentous growth (Fein et al. 1984) on molasses and nor did the experiments show extensive levan or fructo-oligomer formation (Torres and Baratti 1987; Viikari 1988).

Discussion
The presented results demonstrate that Z. mobilis strain U Q M 2716 (ATCC 39676) is not only suited for corn (Doelle et al. 1989), milo (Millichip and Doelle 1989), potato (Richards and Doelle 1989) and sugar-cane syrup (Doelle and Doelle 1989), but also for different types of molasses fermentation to industrial ethanol. Whereas sugar-cane syrup required the addition of nutrients (Doelle and Doelle 1989), B- and C-molasses had no such additional requirement, in contrast to earlier reports (van Vuuren and Meyer 1982). At all molasses concentrations used, sucrose hydrolysis was always complete, in contrast to earlier reports with different Z. mobilis strains ( M u r p h y 1988a-c). It was, however, discovered that one of the reasons for molasses inhibition of fermentation at concentrations higher than 250 g/1 (equal to a 1 : 3 dilution) m a y be inhibition o f some molasses c o m p o n e n t on fructose uptake into the cell or fructose conversion. Such an inhibition could be due to an effect on m e m b r a n e transport or either o f the two enzymes, fructokinase or p h o s p h o glucoisomerase, since glucose conversion and sucrose hydrolysis were not affected. The observations m a d e b y Rhee et al. (1984) could therefore be due to an effect o f ions on fructose utilization. Further work in this direction is in progress. The results also confirmed earlier reports that by using the optimal temperature (Lyness and Doelle 1980) and p H (Lyness and Doelle 1981) sucrose hydrolysis is i n d e p e n d e n t of m o n o m e r i c sugar uptake and that the activity of levansucrase (Lyness and Doelle 1983) is directly related to sucrose concentration (Lyhess and Doelle 1981), provided that glucose does not accumulate (Blackbeard and Doelle 1983). Glucose accumulation not only inhibits sucrose hydroylsis (Blackbeard and Doelle 1983), but also leads to sorbitol formation (Viikari 1988). In c o m p a r i n g molasses fermentation using Saccharomyces species with Z. mobilis, the latter appears to have an advantage in conversion efficiency (Rose 1976), but is vastly superior in regard to fermentation time (Patil and Patil 1989). Zyrnomonas mobilis does not require any nutrient addition, in contrast to yeast (Wolniewizc et al. 1988; Patil et al. 1986, 1989). Higher cell biomass of Z. mobilis obtained through use of flocculant or immobilized strains appears not to increase

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