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close this bookBioconversion of Organic Residues for Rural Communities (UNU, 1979)
close this folderProduction of single-cell protein from cellulose
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Experimental results

The investigations carried out to date are as follows:

a. studies on bacterial fermentation of cellulose using Cellulomonas as the main organism;

b. Aspergillus terreus as SCP from cellulose;

c. growth of yeast on cellulosic substrates.

Studies on Bacterial Degradation of Cellulose

The primary objective in all earlier investigations was to optimize the growth environments of Cellulomonas in such a manner as to increase the rate of degradation of alkalis treated cellulose, and thereby increase the productivity (biomass produced per unit volume per unit time). The problem was investigated in three different sets of experiments: (i) symbiotic growth of Cellulomonas with organisms having the ability to grow on cellobiose as the sole carbon source; (ii) mutation of the parent strain of Cellulomonas by chemical mutagens, and isolation of strains with less fastidious requirements for growth, and (iii) studies on the physiology of the organism in continuous culture with a view towards making the best use of the constituents in the nutrient medium to promote maximum growth. Based upon observations from studies of continuous culture, a simple technique known as gradient feed was developed for growing cells at high densities in batch cultures in laboratory fermenters (9). The experimental results obtained so far from batch fermentations of Cellulomonas are summarized in Table 3.

All cellulose substrates were treated with NaOH** Fermentations were carried out at controlled pH at 6.8 in New Brunswick 7-litre fermenter at 35 C.

Investigations on continuous cultivation of Cellulomonas were conducted with glucose as the carbon source. Although our main interest is in growth of the organism on cellulose, glucose was initially chosen as the substrate in order to understand the basic physiology of the organism, which is more easily observed using glucose. During these experiments, we noted that trace elements in the medium play an important role in regulating the growth rate of the organism. The minimum concentration of Zn(++) ion required for maintenance of steady states at different dilution rates was examined. Moreover, the macromolecular composition of the organism at different dilution rates was determined (Figures 2, 3). As seen in Figure 3, RNA content in the cell is lower in Zn(++). This is indeed an interesting observation, for this may provide a method of decreasing the total nucleic acid content of the cell.



Figure. 2. Variation of the Dilution Rates Dependent upon the Minimum Concentrations of Zn(++) in the Influent for the Maintenance of Constant Biomass at Steady State during Cultivation of Cellulomonas in a Chemostat Dilution Rate



Figure. 3. Macromolecular Composition of Cells of Cellulomonas in a Chemostat under Conditions of Minimum and Excess Amounts of Zn(++) in the Nutrient Feed

Studies of Cellulose Degradation by Asporgillus terreus

Microfungi have attracted but little attention as single-cell proteins for two reasons: (i) fungi are supposedly slow-growing, and (ii) they may produce mycotoxins during cultivation. However, they have several advantages. They can be grown at low pHs and thereby minimize the problem of contamination. Because of the size of the organisms, they may be more economically harvested out of the fermentation menstruum. A number of fungi have growth rates exceeding 0.20 hr(-1) sufficiently fast enough for consideration for the production of SCP (10). Gray has shown that filamentous fungi have been used widely by man either directly or indirectly for food (11). Mycotoxins are produced only by a few organisms in stationary phase as secondary metabolites, and they are synthesized only at a particular stage in the life cycle of the organism. Hence, even in fermentations with such organisms, it may be possible to inhibit the production of toxins by growing the organism at fast growth rates. We have, therefore, attempted to develop a fungal fermentation on cellulose.

We have screened for several fungi and isolated a cellulolytic strain of Aspergillus terreus for further study. Preliminary experiments showed that the organism has the potential to grow on a variety of carbon substrates such as glucose, lactose, cellobiose, and starch as well as on cellulose. Although the germination of the spores required additional growth factors from yeast extract, mycelial growth occurred on carbon substrates in a simple minerals medium. The organism exhibited growth over a wide range of pHs (3.5 to 7.0) and temperatures (30° to 45°C).

A typical experiment on the growth of Aspergillus terreus on different cellulosics is presented in Table 4. The fermentations were conducted in New Brunswick equipment fitted with 7-litre vessels with a working volume of 5 i. The temperature was maintained at 35 C, and pH was kept constant at 3.5 with an automatic pH controller. No undue precautions were taken with regard to conditions of sterility. The mass doubling time at the initial phase of fermentation was approximately 5.5 - 6 hours. The final product of fermentation consisted of filamentous fungi and undigested cellulose. Very few contaminating organisms, predominantly yeast, were detectable in the product. Attempts were made to grow the organism on treated bagasse (2 per cent) in a 100-litre fermenter under non-sterile conditions as batch fermentations. The final product contained between 20 - 22 per cent protein. All our efforts to run the fermentation as a semi-continuous operation proved futile because of contamination.

TABLE 4. Growth of Aspergillus terreus on Cellulose

Expt. Carbon source Inoculum size g/l Biomass (after 20 hr)g/l
1 Solka-floc 1.5 9,0
2 Bagasse 1.2 8.0
3 Bagasse pith 0.8 5.2

The experiments were conducted in 5-litre volume with a substrate loading of 3% at a temperature of 35 C. The pH was kept constant at 3.5. The fermentation was carried out under non-aseptic conditions.

A series of experiments on the continuous cultivation of Aspergillus terreus were conducted using treated solka-floc as the carbon source. A 5-litre working volume was used in these experiments, which were carried out under aseptic conditions. Because of the difficulty of pumping a slurry of cellulose continuously, a substrate level of only 0.15 per cent was used. The experiments were run at different temperatures and pHs. Steady state values were maintained for at least eight residence times before samples were taken for analyses. The results obtained from a few such experiments are summarized in Table 5.

TABLE 5. Continuous Cultivation of Aspergillus terreus on Alkali-Treated Solka-Floc

Expt. Dilution

rate

hr(-1)

Temper-

ature

°C

Dry wt.

of

biomass

mg/l

True

protein

content

mg/l

Residual

cellulose

mg/l

Percent

utilization

of

cellulose

1 0.1 35 868 214 330 78
2 0.11 35 870 196 250 83
3 0.14 40 780 191 270 82
4 0.14 44 737 180 200 87

The experiments were run with a substrate concentration of 0. 15% cellulose The pH was maintained at 3.8 with an automatic pH controller, working volume 51.

As seen in the table, 80 - 85 per cent of the cellulose substrate was assimilated on a continuous basis with a productivity of 103 mg/l/hr when the substrate loading was kept constant at 0.15 per cent. On a commercial scale, substrates may be introduced into the fermenter at a level of 5 per cent. If we allow the luxury of extrapolating, at the present state of the art, a productivity of 3.4 g/l/hr might be achieved. An economic estimate of the process has been made with the following assumptions: 300 days operation of a plant volume of 40,000-litre capacity producing 800 metric tons of product per year containing 25 - 30 per cent crude protein from bagasse. The unit cost of production is approximated at US$180/ton.

The next phase in the development of the project requires pilot studies in an industrial site to solve the problems of scaling up and testing the product for its usefulness as animal feed.

Experiments on the Degradation of Cellulose by Yeast

There are only a few investigations on the degradation of cellulose by yeasts reported in the literature (12, 13). After having successfully completed the experiments on Aspergillus, it was of interest to study the growth of yeasts on cellulose, based upon the knowledge gained during the investigations on the growth of Cellulomonas as well as Aspergillus. A cellulolytic yeast was isolated from piles of sugar-cane bagasse and tentatively identified as a strain of Trichosporon cutaneum. The organism had the ability to grow on several carbon sources, and Table 6 presents the specific growth rates of the organism on different substrates. An 0.5 per cent carbon source was used in all of these experiments. The pH was initially adjusted to 7.0 and the temperature was kept constant at 35 C. Turbidity was measured at intervals with a Klett-Summerson photo-electric calorimeter, and the maximum specific growth rates were calculated from the kinetics of growth.

TABLE 6. Specific Growth Rates of Trichosporon cutaneum on Different Carbon Sources

Expt. Carbon source

(0.5% w/v)

Sp. growth rate
1 Glucose 1.06
2 Cellobiose 1.00
3 Lactose 1.03
4 Maltose 0.81
5 Carboxymethyl cellulose 0.76

Similar experiments were carried out to determine the characteristics of growth of the organism on carboxymethyl cellolose at different temperatures and pHs. In the experiments on the effect of pHs on the growth of yeast, only the initial pH was established and no attempts were made to control the pH during the course of the experiments, because they were only short term and the measurements were completed over a period of six to eight hours. The organism grows well at a temperature between 30 - 40 °C, and optimally at about 35°C. Figure 4 presents the specific growth rates at different pHs. Growth of Trichosporon cutaneum on treated cellulose was fairly rapid. However, during the period of growth, cellulase was bound to the cells during the rapid growth phase, and no detectable amount of the enzyme was found in the supernatant fluid after harvesting the cells from the culture. Figure 5 illustrates the growth of, and cellulose production by, the organism grown on 0.5 per cent treated cellulase in batch culture at 35°C. Cellulase activity was determined by measuring the reducing sugar released from 0.5 per cent CMC incubated with 10 ml of the supernatant fluid or cells from 10 ml of the culture. During the incubation of the cells for measurement of cellulase activity, 10 µg/ml of cyclohexamide was used to inhibit further enzyme synthesis.



Figure. 4. Effect of pH on the Specific Growth Rate of Trichosporon cutaneum Grown on Carboxymethyl Cellulose as the Carbon Source



Figure. 5. Growth of Trichosporon cutaneum and Secretion of Cellulase by the Organism Grown on Treated Solka-Floc

TABLE 7. Solubilization of Different Cellulose Substrates by Trichosporon cutaneum

  Percent solubilization after 72 hrs
Carbon substrates Alkali-treated Non-treated
Solka-floc 64.3 28.6
Rice husk 44.4 22.2
Newsprint 33.3 6.6
Sisal fibres 44.4 40.7
Bagasse pith 38.5 30.7
Bagasse fibres 50.0 32.5

The medium contained 0.5% of cellulose at the time of inoculation. The organisms were cultivated in 250 ml flasks containing 50 ml of medium. The cultures were aerated on a gyrotary shaker kept at a constant temperature of 35°C.

Table 7 shows the ability of the organism to solubilize cellulosic substrates. After 72 hours of growth of the organism on different substrates, the residual cellulose was determined by the method of Updegraff (14). Per-cent utilization was calculated by the formula



where CI = initial amount of cellulose in the medium, and

CF = residual cellulose left after 72 hours of growth.

Studies on the continuous cultivation of the organism on CMC are in progress to determine the kinds and amounts of nutrients required for maximum productivity.