Cultivation Conditions and Nutrient Requirements

2.2.1 Carbon Source

Since, substrate represents a major cost item (estimated to be anything from 30 to 70%), waste will not be tolerated if the process is to have a chance to be economically viable. Since d-xylose is a major component of cellulosic biomass and since few organisms can convert it at all—and those who do usually do so inefficiently, the cellulolytic strains that can convert d-xylose to ethanol represent a great opportunity (Schneider 1989), and they should be scrutinized further in future studies. Especially notable are the cellulolytic strains of Monilia, which are capable of producing ethanol from both d-glucose and d-xylose. In a study, this fungus converted more than 40% of d-xylose into ethanol in 7 days (Kumar

Table 1 Fungi responsible for cellulose degradation

Organism

Features

F. oxysporum

F3

Direct conversion of cellulose into

ethanol

F. oxysporum

DSM 841

Efficient direct conversion of waste

cellulose to acetic acid

Monilia sp.

Direct conversion of cellulose to

ethanol

N. crassa

Direct conversion of

cellulose/hemicellulose into

ethanol

et al. 1991). Like Monilia, the fungus F. oxysporum also successfully converts a variety of carbon sources: glucose, xylose, cellobiose, with a yield of 5.0-9.1 g/l of ethanol. N. crassa is yet another species that biologically transforms cellulose all the way to ethanol (Singh et al. 1992). It should be noted that many fungal cellulose degradation studies and fungal cellulase production studies employ well-defined model substrates such as carboxymethyl cellulose (CMC), Avicel (which is more crystalline), or xylan. The synthetic media are further supplemented with minerals and yeast extract. Although defined media tend to yield more reproducible laboratory results, they do not realistically approximate commercial processes. On a related point, these model compounds also serve as substrates for standardized cellulase activity measurements. For example, a common gauge of endoglucanase activity is decreased CMC viscosity. Note that a decrease in the CMC viscosity does not linearly correspond to an increase in the reducing sugar level.

2.2.2 Nitrogen Source

Nitrogen is a major cellular constituent of any organism, and fungi are certainly no exception. Any successful media formulation must carefully balance microorganism's inherent need for nitrogen. Nitrogen limitation usually leads to slow or no growth or even death of fungi, slow xylose consumption, and retarded ethanol production. Table 2 summarizes the different sources of nitrogen and the typical concentration levels present in fungal growth media in a laboratory setting. In a defined media formulation, typical nitrogen sources are ammonia, nitrates, and urea. In complex media, peptone, tryptone, yeast extract, wheat bran, and an array of protein digests supply other crucial but less understood set of nutrients. Metabolic pathways trace the fate of carbon rather than nitrogen sources. Nonetheless, nitrogen sources' effect on fungal metabolism can be pronounced. For example, Sale (1967) reports that ammonium ions stimulate glycolysis by counteracting ATP's inhibition of phosphofructokinase, and they stimulate pentose phosphate pathway by derepressing glucose-6-phosphate dehydrogenase. However, the effects of nitrogen sources are generally not as well understood. From

Table 2 Nitrogen sources in lignocellulosic conversion processes by fungi

Nitrogenous compounds Concentration (g/l)

Ammonium nitrate 1.0

Ammonium dihydrogen phosphate 2.0

Potassium nitrate 2.5

Sodium nitrate 2.0

Urea 2.0

Peptone 5.0

Yeast extract 0.25

Potato protein liquor 40

economic feasibility viewpoint, supplementing nitrogen from complex sources is undesirable because the practice adds significantly to the cost. A future challenge is to eliminate completely the need for nitrogen addition and to satisfy fungi's nitrogen demand entirely from natural lignocellulosic sources. At least, one would consider cheaper sources of nitrogen.

2.2.3 Minerals and Vitamins

Minerals, trace elements, vitamins, and growth factors play a vital role in the growth of fungi and their biosynthesis of metabolites. There has been no systematic study on the effect of minerals and trace elements such as Ca, Mg, Fe, Zn, Cu, Co, and Mn on growth of fungi and ethanol production by fungi. The present strategy is to provide sufficient quantity of these compounds, estimated based on information gathered from other microbial fermentation, such that they do not become rate limiting. However, it is unclear what constitutes an optimal level. The large number of compounds involved immediately translates to unpractical number of runs. For example, if one were to conduct brute-force experiments by varying one variable at a time, say, each variable set at three separate levels (high, medium, and low), n variables yields 3n combinations if the variables interact with one another, or 3n combinations if the variables do not interact. To appreciate the magnitude of this undertaking, 10 trace compounds translates to 310 = ~300,000 possible combinations! And that does not count the unsuccessful runs. Although a sound experimental design can reduce the number of runs, the number of runs required remains utterly unpractical if the variables interact.

2.2.4 Aeration

Under aerobic conditions, the fungi listed in Table 1 grow well but are unable to produce ethanol. On the other hand, under anaerobic or microaerobic conditions, they are able to produce ethanol but grow only slowly. Thus, if one is to pursue cellulose-to-ethanol with a single fungal species, a common strategy is to cultivate fungi first under aerobic conditions, followed by ethanol production under anaerobic conditions. For example, in a study utilizing N. crassa, the mold grew first aerobically for 48 h. Subsequently, the content of the growth flask was transferred to a special flask that had a capillary opening at the top to exclude oxygen from entering while allowing carbon dioxide to escape as ethanol is produced (Singh et al. 1992).

Compared to higher organisms, fungi are more susceptible to changes in their surrounding environments. However, the cytoplasmic pH of the fungi tends to change very little over a wide range of extracellular pH. In most prior studies, which have been conducted without active pH control, the initial pH values are within the range of 5-6. Both the rate of cellulose degradation and product distribution depend on the pH. For example, Enari and Shihko (1984) report an optimal pH of 5.5 for ethanol production for F. oxysporum, but acetic acid production increased considerably at pH 6.0. For N. crassa, maximum ethanol production occurred in the range of 5.0-6.0, which coincided with maximum cellulase activity (Deshpande et al. 1986).

In nature, fungi may be exposed to different environmental conditions that can inhibit their growth. In general, the intrinsic enzyme (including cellulase) reaction rate and its deactivation rate both increase with temperature. As temperature increases, the rate of enzyme deactivation surpasses that of enzyme reaction temperature; thus, the apparent enzyme reaction rate exhibits a maximum with respect to temperature. Deshpande et al. (1986) show that N. crassa works best at 37°C. It converts more than 90% of cellulose into ethanol within 4 days at 37°C. However, above this optimum, the cellulase activity increases but the production of end product, such as ethanol, decreases. Table 3 shows optimum pH and temperature needed for the conversion of cellulose by different fungi.

Light plays an important role in the growth of filamentous fungi. It can hinder or arouse the emergence of mycelia, and sensitivity to light is species dependent. In Trichoderma, the most efficient wavelengths to induce growth are 310, 430, 455, and 480 nm.

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