徐峻宇

【Abstract】Simultaneous saccharification and fermentation (SSF) of waste newspapers constitutes an attractive process for ethanol production from biomass. After a deinking process, the paper sludge undergoes simultaneously an enzymatic hydrolysis (the breakdown of cellulose into glucose by cellulase) and yeast fermentation (the concentration of glucose into ethanol). This method is applicable on waste newspapers and even on other waste materials containing cellulose. Compared with other methods, SSF is considered by many scientists to be the most efficient technique for producing ethanol from cellulose because it gives the highest yield of ethanol, is less time consuming and has a low cost.

【Keywords】Simultaneous saccharification and fermentation (SSF) cellulose cellulase waste newspaper yeast.

1 Introduction

At least 125 million metric tons of municipal solid wastes are disposed of annually in landfills in China. Paper and paper products make up about 30% of the municipal waste. The recycled fibers from paper related products have repeatedly undergone repulping treatment. The short fibers generated in the waste stream were chemically swelled and became much hydrated. This causes a very high water holding capacity of the waste known as recycled paper sludge (RPS). One of such potential products from RPS is ethanol. As the similar structure, The RPS, starch, waste newspapers etc could be the raw materials for producing bio-ethanol.

Currently, one of the most investigated processes to produce bio-ethanol from starch and cellulose sources is called simultaneous saccharification and fermentation (SSF). The SSF process, which was first used in 1977, gives high rates and yields for ethanol production from biomass, when the organic substrates hydrolyzed by enzymes into glucose are coupled with yeast fermentation in one reactor (Mohagheghi et al. 1991). Because the hydrolysis of cellulose and the fermentation step are processing in the same equipment continuously, the result of hydrolysis, glucose, can be utilized by the yeast fermentation. It avoids the inhibition of high yield of glucose to the cellulase hydrolysis. There is only one step in SSF, and the equipments are simplified, the energy sources and the total production time are saved.

2 Materials and mathods

2.1The raw materials in Simultaneous Saccharification and Fermentation (SSF)In general, many types of substances can be treated as raw materials for producing ethanol. Traditionally, starch is the most important material. But for the economical thinking and saving foodstuff, cheap materials, such as industrial wastes, have to be used. Compared with the starch, they reduce the production cost of the production. The most important common ground is that the raw materials must contain cellulose. For example, softwood, waste paper sludge and straw all contain cellulose. In this experiment, waste newspapers are used as raw material.

2.2 Simultaneous Saccharification and

Fermentation

To operate an SSF system, an optimum temperature is required with respect to the saccharification enzymes, which are activated between 45-55 C. Therefore, research on thermophilic microorganisms has been steadily increasing because they are capable of growth up to 49 C and capable of producing alcohol at and above 40 C (Banat et al. 1992). Some yeasts of the genus Kluyveromyces were found to be more thermotolerant than Saccharomyces or Candida strains (Hacking et al. 1984).

SSF has the economical advantage compared with separate hydrolysis and fermentation (SHF). But the critical problem with SSF is the difference in temperature optima of the cellulases and the fermenting microorganisms. Saccharomyces strains are well known as good ethanol producing microorganisms; however they require an operating temperature of 35 C. Fungal cellulases, which are most frequently applied in the cellulose hydrolysis, have an optimum temperature of 50 C. At lower temperatures, the substantially lower hydrolysis rates would be unfavourable in terms of increased processing time. A possible solution to solve this problem is using thermotolerant yeast strains instead of Saccharomyces strains, which would allow higher processing temperatures, thus increased rates of the hydrolysis. In the experiment, the Temperature Tolerant Active Dry Yeast was selected, and the temperature was 40 C.

From primary experiments of different authors (Wyman et al. 1992; Sosulski & Swerhone 1993), it became obvious that a high degree of hydrolysis of paper products to glucose requires low substrate concentration, prolonged hydrolysis time and high enzyme loading. High enzyme concentration can increase conversion rates, but the enzyme cost is too high. Low concentrations of substrate would increase the capital cost of equipment and would yield low concentrations of sugars for fermentation and ethanol for distillation (Sosulski & Swerhone 1993).

The experimental results of enzymatic hydrolysis at 5 and 7.5% (w/v) initial substrate concentration and 15 and 45 FPU/g (1FPU/g means 1g cellulase makes 1mg reducing sugar in 1 hour at 50 C) substrate of enzyme loading are shown. Above 7.5% (w/v) of initial substrate concentration, it was difficult to keep the substrate in suspension, so higher concentrations were not used. Comparable rates at different substrate and enzyme loading were obtained. Enzymatic hydrolysis yields (expressed as a percentage; the glucose produced in the enzymatic hydrolysis divided by the potential glucose in the substrate) for conditions assayed were in the range of 43.8- 47.9 %.( M. Ballesteros et al, 2002)

Final ethanol yields (72 h) obtained in SSF experiments are shown. SSF yields are expressed as percent of theoretical yield. In standard SSF assays, initial substrate concentrations above 10% (w/v) were unsuitable due to viscosity of mixtures. As expected, high enzyme loading and low substrate concentration increased SSF yields.

The amounts of ethanol produced at 5% (w/v) substrate loading were equivalent to 74.2 and 80.3% of the available glucose at 15 and 45 FPU/g of substrate, respectively. These results show that the amounts of cellulose converted to glucose in SSF process are remarkably higher than those obtained in enzymatic assays. The ethanol yield in a typically fermentation process with K. marxianus CECT 10875 is about 0.45 g ethanol/g glucose (Ballesteros 2001). Thus, the ethanol concentration that could be obtained in a separate hydrolysis and fermentation process would be about 4.4 and 6.5 g/l at 15 and 45 FPU/g of substrate, respectively. The higher ethanol concentrations obtained in SSF (8.2 8.9 g/l) reveal enzymatic hydrolysis yields in the range of 85 90% in that process, demonstrating its advantage over the separate saccharification and fermentation process.

The highest ethanol yield was obtained at 5% (w/v) substrate concentration and 45 FPU/g of substrate, but this enzyme loading can be considered too expensive for the SSF process. In fact, lower cellulase loading of about 10 FPU/g of substrate has been reported for other paper-derived materials as recycled paper sludge (Lark et al. 1997) and primary clarified sludge (Duff et al. 1994).

3 Results and discussion

In the experiments, the substrate loading value was set in a normal range: 10g paper sludge was added into 100 mL. After adding the other ingredients, the final volume was about 160 mL. Therefore, the loading value was 10g/160mL = 62.5 g/L.

In the experiment with the Danbaoli TTADY and a cellulase concentration of 40FPU/g, 10 ml of sample was taken each time for determining the yield of glucose. But in the following experiments, only 0.1 ml of sample was taken. This was done because, in the first experiment, there is only a little glucose and much more glucose was tested in the next two experiments; 10 ml contains too much glucose and cannot be measured.

After the first experiments with the Danbaoli Yeast and a cellulase concentration of 40FPU/g, concentration of cellulase was lowered to 20 FPU/g.This was done because the cost of cellulase was considered in. A high enzyme loading gives a high ethanol yield, but 40 FPU/g of cellulase can be considered too expensive for the SSF process. If the first experiment had succeeded, a higher amount of ethanol would have been produced. Lower cellulase loading of about 10 FPU/g of substrate has been reported for other paper-derived materials as recycled paper sludge (Lark et al. 1997) and primary clarified sludge (Duff et al. 1994). Anyway the lower cellulase loading was chosen instead of 40 FPU/g.

The experimental data show that the glucose concentration increases with the time. After 65 hours or 90 hours, the concentration was getting a bit lower. The concentration of glucose was not getting lower rapidly, but only after many hours, and it only decreases a little. The addition of Tween-20 increases the glucose concentration 7.7% - 9.8%.

It also show that the ethanol yield increases, but after about 90 hours, the ethanol concentration remains constant. This is similar with the results obtained by some scientists such as Malek Alkasrawi et al. in 2003. As expected, the ethanol yield increases after the addition of Tween-20.

Related with the amount of ethanol and glucose, the reason why glucose was not consumed soon can be found. The most possible reason is that the transformation of cellulose to glucose is perfect, but the transfer rate of glucose into ethanol is not so high. In other words, the hydrolysis rate is larger than the fermentation rate in most of time. Until 65 to 90 hours passed, the fermentation rate exceeds the hydrolysis rate so that the concentration of the glucose goes down. To get better results, it is suggested that a lower amount of paper sludge should be chosen in the further research. If the glucose were not consumed fast enough, the redundant of glucose would inhibit the reaction of cellulose hydrolysis. The transfer rate and the final yield of ethanol are both influenced. In addition, because the transfer rate of hydrolysis is much higher than fermentation rate, a lower concentration of cellulase can also be tested in further research in order to fit a suitable condition in SSF.

Figure 1.1 The influence of Tween-20 to the yield of ethanol with Angel Yeast and Danbaoli Yeast ([cellulase]=20FPU/g).

In the experiment with the Danbaoli Yeast, the glucose concentration is getting too high at 89 hours with Tween-20. But it may be a test error in the experiment. In the two kinds of yeast are compared, it can be concluded that the ethanol yields with and without Tween-20 for Danbaoli yeast are both a little higher (3.9% with Tween-20, 6.0% without Tween-20) than for Angel yeast. It proves that both of the yeasts have high fermentation ability, but Danbaoli yeast is a bit more excellent than the other.

4 Conclusions

The main objectives of the present study were to compare the performance of two different thermotolerant yeasts in the SSF, and the efficiency of Tween-20 addition for improving the process. The results showed that the two yeasts have a similar capability in the fermentation process, but the Danbaoli Temperature Tolerant Yeast is a bit more efficient than the other. It is obvious that the yield of ethanol is higher with Tween-20 addition than without Tween-20 addition. Some advantages however are still not shown in the experiment such as reducing the time to reach the maximum yield of ethanol by Tween-20.

In fact, a very low amount of glucose should exist in the SSF process after several hours, but this phenomenon did not appear in the experiment, because the substrate was too much more and yeast could not digest all glucose immediately. The high glucose existing would influence the reaction speed of enzymatic hydrolysis and the time to reach the highest amount, and the final yield of ethanol.

In further research, other aspects of the experiments could be examined. The SSF condition temperature is 40 C and it seems suitable for both cellulase and yeast activity. The temperature could be adjusted a bit (for example 38 C, or 41 C) to compare if it is better. In the experiment, there was redundant glucose left and it influenced the results. The addition of less paper sludge or less cellulase concentration should be considered to solve this problem. Also for reducing the costs, 10 and 15 FPU/g of cellulase can be tested and the most economical one can be selected. After some hours, the ethanol concentration steps increasing. So the appropriate time for stopping the SSF process can be tested, because it is significative for the industrial use of the SSF process.Reference:

[1]Malek Alkasrawi, et al., 2003.The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol.

[2]Nicole Lark, et al., 1996. Production of ethanol from recycled paper sludge using cellulase and yeast, Kluveromyces marxianus.

[3]Hetti Palonen, 2004. Role of lignin in the enzymatic hydrolysis of lignocellulose.