Progressive Gardening

Products And Effects

Bioconversion of distillery waste by means of fungi brings a double benefit: the effluent is substantially purified and, in addition, it is possible to obtain useful products, such as protein-rich fungal biomass, ethanol, enzymes, etc. Results of bioconversion by yeasts are given in Table 2.

In most experiments C. utilis was used as the chosen microorganism, especially for cane molasses stillage (Friedrich et al. 1992). The quantity of cells produced varied significantly with the concentration of the substrate and with the addition of N sources. With increasing nitrogen supplementation a concentration of biomass of up to 25 g/l could be obtained with C. utilis in cane molasses stillage (Cabib et al. 1983). The high quantity of C. utilis biomass of 22 g/l obtained by Wang et al. (1980) in continuous fermentation was a result of cell recycling. Crude protein content in the biomass of Candida utilis accounted for 2858% of the cell mass. With the same species the maximal COD reduction in the effluent was 52% (Nudel et al. 1987) and the highest BOD reduction was 55% (Matsuo et al. 1965).

Among yeasts shown in Table 2, other than C. utilis, outstanding biomass concentration of 70 g/l was produced by S. cerevisiae during continuous cultivation in enriched rice spirit stillage (Yang and Tung 1996). In general, concentrations of about 20 g/l were relatively high. The biomass of Candida brumptii showed the highest protein content of 53%. Kluyveromyces marxianus seemed to be outstandingly efficient, considering the COD reduction of 60-70%, while its biomass concentration of 10 g/l was low in comparison with C. utilis. The results of reduced COD and BOD are given in terms of relative values and the absolute reduction is dependent on the initial value.

Filamentous fungi are cultivated mostly in batch processes. Results of bioconversion of distillery waste by filamentous fungi are given in Table 3.

When compared with yeasts, filamentous fungi are more effective in consuming polluting substances, since COD and BOD reductions of over 80 and 90%, respectively, have been recorded. A. oryzae (Araujo et al. 1977) and G. deliquescens (Rolz et al. 1975) in sugarcane stillage, Penicillium strains in wine vinasse (Magny et al. 1977), and G. candidum in malt whisky stillage (Quinn and Marchant 1980) proved to be the most promising in this respect. The highest amount of biomass was reached with A. awamori var. kawachi in rice-shochu distillery wastewater (Morimura et al. 1994a). It was observed that on average the protein content in the biomass of filamentous fungi was lower than that of yeasts. G. candidum, however, was found to produce the highest protein content; there was 45.5% "true" protein in the biomass as determined by the biuret method (Quinn and Marchant 1980).

An overview of the bioconversion products and effects using mixed cultures of different microorganisms is given in Table 4.

In general, mixed cultures were the most effective in substrate utilization, based on COD values. A co-culture of 15 yeast strains in a high-loaded beet molasses stillage of initial 74.5 g/l COD, produced the highest amount of biomass, 28.9 g/l, in batch culture. When diluted medium was applied, the biomass yield was much lower (Huyard et al. 1986). A successful approach seems to be a two-step separated cultivation of C. utilis and P. varioti by which a total amount of 22 g/l cell material and a COD reduction of as much as 90% were obtained. During the first step, C. utilis was used mainly for SCP production, whereas P. varioti in the second step consumed the reducing organic substances in the liquid phase (Azzam and Heikel 1989; Bottaro-Castilla et al. 1984). Similar results were observed in a two-step continuous cultivation of C. utilis and A. niger grown separately (Nudel

Table 3 Products and effects of bioconversion of distillery waste by filamentous fungi

		Biomass	Protein	COD reduction	BOD reduction
Fungus	Stillage	(g/L)	(%)	(%)	(%)	References
A. campestris	CMS	13	45			Falanghe (1962)
A. fusidioides	CMS	11	40			Rosalem et al. (1985)
A. niger	CMS	8-13	30-40	46-78		De Lamo and De Menezes (1978) and Rosalem et al. (1985)
A. awamori var.	Rice	40	40		76 (TOC)	Morimura et al. (1994b)
kawachi
A. niger	Fruit	4-20	12-36	50-70		Friedrich et al. (1983, 1986) and Gunde-Cimerman et al. (1986)
A. oryzae	CMS	14-17	35-50	61 -88	79-83	AraUjo et al. (1977)
A. oryzae	CMS	12-15	39	48-72	78	De Lamo and De Menezes (1978)
A. phoenicis	CMS (rum)	20	21		58	De Gonzales and De Murphy (1979)
G. candidum	Whiskya	3.5		80.6	92	Quinn and Marchant (1980)
G. candidum	Whisky	11-27		32-78	63-91	Quinn and Marchant (1980)
G. candidum	Whiskyb	34	46		87	Quinn and Marchant (1980)
G. deliquescens	CMS (rum)	11-19		60-85		Rolz et al. (1975)
M. verrucaria	CMS (rum)	11-12		79-82		Rolz et al. (1975)
P. elegans	CMS (rum)	11-14		62-66		Rolz et al. (1975)
P. varioti	CMS	13- 25	40	43 -70		Bottaro Castilla et al. (1984), Cabib et al. (1983), and Nudel et al. (1987)
P. varioti	CMS	5		70		Azzam and Heikel (1989)
P. oxalicum	Raisins	12	34			Aran (1977) and Yazicioglu et al. (1980)
Penicillium sp.	CMS (rum)	12-16		65		Rolz et al. (1975)
Penicillium spp.	WV	13	41		91	Magny et al. (1977)
T. viride	CMS	12- 28		59-79		Nudel et al. (1987) and Rolz et al. (1975)

CMS, cane molasses stillage; WV, wine vinasse. a Diluted five times. b Continuous, two stage.

CMS, cane molasses stillage; WV, wine vinasse. a Diluted five times. b Continuous, two stage.

Table 4 Bioconversion of distillery waste by mixed microbial cultures

Microorganisms	Stillage	Biomass (g/L)	COD reduction (%)	Cultivation mode	References
P. varioti + T. viride	CMS	12-17	50-64	Batch	Nudel et al. (1987)
T. viride + A. oryzae	CMS	10-13	50-54	Batch	Nudel et al. (1987)
C. utilis + P. varioti	CMS	8-12	45-50	Batch	Nudel et al. (1987)
C. utilis + C. acetoacidophilum	CMS	16-17	65	Batch	Nudel et al. (1987)
C. utilis + B. flavum	CMS	16	65	Batch	Nudel et al. (1987)
Azotobacter + C. utilis	CMS	16		Batch	Nudel et al. (1987)
C. utilis + A. niger	CMS	16-17	89	Cont. serial	Nudel et al. (1987)
C. utilis + P. varioti	CMS	22	92	Batch	Bottaro Castilla et al. (1984)
C. utilis + P. varioti	MS	22	90	Batch, two step	Azzam and Heikel (1989)
C. utilis + P. varioti	CMS	14-16	85	Cont. serial	Bottaro Castilla et al. (1984)
15 yeasts	BMS dil.	7	79	Batch	Huyard et al. (1986)
15 yeasts	BMS dil.	9	72	Cont.	Huyard et al. (1986)
15 yeasts	BMS	29	74	Batch	Huyard et al. (1986)
13 yeasts	BMS dil.	7-12	68-75	Cont.	Malnou et al. (1987)
G. candidum + C. crusei + H. anomala	MWS	13	55	Batch	Barker et al. (1982)
G. candidum + C. crusei + H. anomala	MWS	5	32	Cont.	Barker et al. (1982)
A. awamori + T. reesei	Apple dil.	5	31	Batch	Friedrich et al. (1987)

CMS, cane molasses stilläge; BMS, beet molasses stillage; MS, molasses stillage (unknown origin); dil, diluted; MWS, malt whisky stillage.

CMS, cane molasses stilläge; BMS, beet molasses stillage; MS, molasses stillage (unknown origin); dil, diluted; MWS, malt whisky stillage.

Table S Bioconversion of stillage by continuous cultivation of fungi

	Dilution rate	Productivity	Biomass	COD reduction	BOD reduction
Fungus	(h"1)	(g/L/h)	(g/L)	(%)	(%)	References
C. utilis	0.383	4.24	17.9		34.6	Wang et al. (1980)
C. utilis	0.365	4.06	21.9		37.1	Wang et al. (1980)
C. utilis	0.22	2.64	12			Cabib et al. (1983)
C. utilis	0.27	3.24	12			Cabib et al. (1983)
C. utilis	0.2	1.8-2.0	9-10	35		Bottaro Castilla et al. (1984) and Nudel et al. (1987)
C. utilis	0.2
P. variotia	0.1	2.3-2.6	19- 21	85		Bottaro Castilla et al. (1984)
C. utilis	0.2
A. nigera	0.1	2.7-3.2	15-17	89		Nudel et al. (1987)
K. marxianus	0.3	3	10-11	60-70		Braun and Meyrath (1981)
G. candidum	0.125	2.24		30	50	Quinn and Marchant (1980)
G. candidum 1st step	0.125
2nd step	0.10		31		50	Quinn and Marchant (1980)
G. candidum 1st step	0.125
2nd step	0.085	1.72	34		87	Quinn and Marchant (1980)
G. candidum + C. crusei +	0.10	0.48	4.8	31.5		Barker et al. (1982)
H. anomala
G. candidum + C. crusei +	0.20	0.36		15.9		Barker et al. (1982)
H. anomala
G. candidum + C. crusei +	0.35	0.42		2.0		Barker et al. (1982)
H. anomala
Hansenula sp.	0.12	~ 1	8.5	35.7		Shojaosadati et al. (1999)
G. deliquescens	0.03		17 - 22	61 -50		Rolz et al. (1975)
S. cerevisiae	0.016		70b			Yang and Tung (1996)
Association of 15 yeasts	0.133-0.134	0.5-1.6	11	68		Huyard et al. (1986) and Malnou et al. (1987)

a Two step process. b Enriched stillage.

a Two step process. b Enriched stillage.

et al. 1987). Summarizing the results of conversion of both steps, 17 g/l biomass and 89% COD reduction were obtained. In general, it was observed that the combination of yeast with filamentous fungi resulted in improvement of COD reduction, and improved wastewater purification.

While the batch cultivation mode is most frequently used, continuous cultivation of fungi in stillages can have some advantages regarding the product yield (Table 5).

As shown in Table 5, an outstanding biomass yield of 70 g/l was produced by S. cerevisiae in enriched stillage from rice spirit distillation when the dilution rate was low (Yang and Tung 1996). On the other hand, dilution rates of over 0.36 h2 with cell recycling resulted in a productivity of over 4g/l/h Candida biomass and a steady-state biomass concentration of 18-22 g/l (Wang et al. 1980). In continuous culture, COD was reduced most effectively by using K. marxianus (Braun and Meyrath 1981). A serial culture of a yeast and a filamentous fungus in two steps gave the most promising results with respect to both biomass yield and efficient consumption of organic matter (Bottaro Castilla et al. 1984; Nudel et al. 1987). Two-step cultivation of G. candidum also seemed to be very suitable, with the steady-state biomass of 31-34 g/l (Quinn and Marchant 1980). The continuous cultivation process could lead to a higher protein content in the biomass and enhance the stability of the culture as well as its resistance to contamination (Barker et al. 1982).

Microbial biomass rich in protein is the main product of fungal bioconversion of stillages. Some information is available about cell material composition. It appears that C. utilis biomass composition is similar to that of other yeasts grown in carbohydrate media. Microbial protein has a good balance of amino acids with the exception of those containing sulfur, such as cysteine and methionine; the content of the latter amino acids is generally low in microbial biomass (Cabib et al. 1983; Quinn and Marchant 1979b; Yazicioglu et al. 1980). However, lysine is in excess when compared to the recommended level of the FAO standards (Quinn and Marchant 1979b). Yeast biomass from vinasse is rich in nitrogen, vitamins, and other biologically active substances. The vitamin B complex of yeast is very efficient and could not be adequately substituted by a mixture of analytically pure vitamins (Cabib et al. 1983; Yazicioglu et al. 1980).

Nutritional evaluation of yeast biomass grown in malt whisky distillery slop showed that it was suitable for nonruminants with a net protein utilization value of 0.40, and a digestibility of 0.67. The biomass was not toxic, as confirmed by toxicological tests (Barker et al. 1982). When molasses stillages were used, K and Mg contents could have laxative effects (Araujo et al. 1977). The nucleic acid content in cell material should be as low as possible; filamentous fungi have an advantage over yeasts in this respect (Araujo et al. 1977). It has been observed that levels of DNA and RNA were lower in batch than in continuous cultures (Quinn and Marchant 1979b). Feeding experiments of Aspergillus biomass in a diet for chicks, demonstrated excellent acceptability, good weight gain, and no toxicity; the biomass had a very good protein efficiency ratio, being comparable to meat and soya meal (Araújo et al. 1977). A 1% methionine addition to Candida yeast biomass increased the biological valúe, which was not much lower than that of casein (Cabib et al. 1983).

With the use of fungi for bioconversion of distillery wastewaters, not only fúngal biomass bút also other prodúcts can be obtained. Secondary ethanol can be produced by the original yeast used in a primary alcohol fermentation, if the distillation is performed at low temperatures in vacuum (Teramoto et al. 1993; Ueda and Teramoto 1995; Ueda et al.

1991). Microbial polysaccharides can be produced, such as pullulan with Aureobasidium sp. (Leathers and Gupta 1994) or chitosan with A. atrospora or G. butleri strains (Yokoi et al. 1998). Hydrolytic enzymes can be produced by filamentous fungi. Cellulases and other glucosidases can be products of bioconversion when cellulolytic fungi are grown in fruit distillery slops (Friedrich et al. 1986; 1987; Gunde-Cimerman et al. 1986). Some ascomycetes, especially A. awamori var. kawachi, are able to produce starch saccharifying enzymes from shochu distillery wastewater (Morimura et al. 1991;

1992). Acid proteases are produced mainly with Aspergillus species in rice (Morimura et al. 1994a; Yang and Lin 1998) and barley (Morimura et al. 1994a) shochu stillages. Possible products are a yeast pigment, astaxanthin, from P. rhodozyma (Fontana et al. 1997) and plant growth hormones, obtained by growing white rot fungi, such as F. trogii or T. versicolor (Yürekli et al. 1999).