|Bioconversion of Organic Residues for Rural Communities (UNU, 1979)|
|Possible applications of enzyme technology in rural areas|
In examining the prospects for a simple process for the degradation and utilization of cellulose, we chose to look at the thermophilic anaerobic bacterium, Clostridium thermocellum. This organism grows well at temperatures of 60 C and above. Most important, it has been observed that this organism has the unique capability to accumulate sugars while degrading and growing on cellulose (10). This phenomenon is illustrated in Figure 2. In this example, medium containing 11 g per litre of solka-floc was inoculated with C. thermocellum. During the course of the fermentation, there is a rapid accumulation of reducing sugars approximately in parallel with growth and cellulose (expressed as CMCase) production From the degradation of approximately 10 g/l of cellulose over the course of 60 hours, there is an accumulation of almost 7 g/l of reducing sugars. Thus, one achieves approximately a 65 per cent yield of reducing sugars from the cellulose that is hydrolyzed
When the sugar products are examined by means of high pressure liquid chromatography (HPLC) (Figure 3), the predominant products are glucose and cellobiose. When C. thermocellum is grown on natural cellulosic materials containing hem-cellulose, there is also an accumulation of xylose and possibly xylobiose. This observation is particularly interesting, because C. thermocellum will not use the pentoses for growth.
We examined both cellulase and xylanase activity in cell-free broths from C. thermocellum grown on a variety of substrates, as shown in Table 2. When C. thermocellum was grown on these substrates, the ratio of xylanase to CMC's activity was consistently one. These results suggest that cellulose activity and xylanase activity are caused by the same enzymes. This observation has been made in other organisms (11), and the dual activity of the cellulase enzymes would account for the simultaneous accumulation of pentoses and hexoses.
TABLE 2. Production of Xylanase and Cellulose Activity by C. thermocellum Grown on Selected Carbon Sources
* The variation in the values among the substrates reflects the time when the fermentations were ended, as well as C. thermocellum's ability to grow and produce enzymes on the particular substrate.
In an attempt to improve and optimize the process, we examined the influence of pH control during the course of the fermentation. In the previous experiments, pH was set initially at 6.8 and then left to fall during the course of the fermentation. Results shown in Table 3 illustrate the difference in performance for C.thermocellum with controlled pH (6.8) and uncontrolled pH fermentations on cellulose. As seen by the results in this table, with pH control there is greater degradation of cellulose, increased cell mass formation, increased synthesis of fermentation products, e.g., ethanol and acetic acid, but markedly less accumulation of reducing sugar. These results suggest that the marked sugar accumulation during growth on cellulose results at least in part from a restriction in growth, probably by the decreased pH, in the presence of excess cellulase capacity. Hence, cellulose is hydrolyzed at a rate faster than it can be utilized.
TABLE 3. Comparison of Results from pH-Controlled and Non-pH-Controlled Fermentations of C thermocellum Grown on Solka-Floc
|Cellulose, g/l||10.1||2 4||10.0||1.8|
|Dry cell weight, g/l||-||0.5||-||0.8|
|Reducing sugar, g/l||0.3||6.0||0.1||2.4|
|Acetic acid, g/l||-||1.0||-||2.7|
The approach illustrated by the above results offers the opportunity for in vivo saccharification of ligno-cellulosic materials to accumulate sugars, as well as the possibility for a direct fermentation for product formation from cellulose. For example, C. thermocellum will produce ethanol, acetic acid, and lactic acid directly from cellulose. Product accumulation is shown in Figure 4, which presents results from C. thermocellum grown on cellulose added intermittently during the course of fermentation. This allowed an increased amount of cellulose to be acided to the broth. A total of 20 9 of cellulose were added, and this resulted in the production of 8.5 g/l of reducing sugars and 4 g/l each of ethanol and acetic acid.
The above example illustrates the direct conversion of cellulose to ethanol. Through genetic manipulation, it is possible to minimize the amount of acetic acid, which is usually produced in molar ratio of 1:1 with ethanol, so that the ethanol-acetic acid ratio becomes 8:1 (12). This is an example of what one can do with high technology to manipulate the ceil in order to generate a cell line with some desired properties, in this case, over-production of ethanol directly from cellulose. One of the bottlenecks in this fermentation is the sensitivity of C. thermocellum to ethanol and aceitic acid. A eel) line that is tolerant to ethanol concentrations of approximately 5V per cent has been developed, and thus we have again modified this cell line to achieve a desired final objective (12).