Utilization of fungi for biotreatment of raw wastewaters

Fungal biomasses are capable of treating metal-contaminated effluents with efficiencies several orders of magnitude superior to activated carbon (F-400) or the industrial resin Dowex-50. Additionally, fungal biomasses are susceptible to engineering improvements and regeneration of their capabilities. With regard to organic pollutants, excessive nutrients and dyes, fungi can remove them from wastewaters, leading to a decrease in their toxicities. However, the detoxification rates seem to be dependent on media and culture conditions. The postreatement by anaerobic bioprocesses of effluents that have been pretreated with fungi can lead to higher biogas than the original effluents. In addition to the degradation of organic pollutants, fungi produce added-value products such as enzymes (LiP, MnP, Lacc, amylase, etc.) and single-cell protein (SCP). Most research on fungal capacities to purify polluted effluents has been performed on a laboratory scale, hence there is a need to extend such research to pilot scale and to apply it to industrial processes.

Phanerochaete chrysosporium is the model of white-rot fungi for the production of peroxidases (Bumpus et al., 1985;Rodriguez et al., 1999).Beyond the production of such relevant metabolites, fungi have been attracting a growing interest for the biotreatment (removal or destruction) of wastewater ingredients such as metals, inorganic nutrients and organic compounds (Akthar and Mohan, 1986;Field et al., 1993;Feijoo and Lema, 1995;Palma et al., 1999;Coulibaly, 2002).
The focus of this review therefore concerns the use of fungi to remove or degrade various wastewater constituents.Some instances of synthetic wastewaters are reported, but only the contributions of fungal biomass in the biological treatment of raw wastewater are discussed in some length.

DOMESTIC SEWAGE
Domestic sewage contains carbon and nutrient sources that can be removed by fungal biomass.In an early investigation, Thanh and Simard (1973) demonstrated the capacities of seventeen fungal biomasses to remove phosphates (84.1%), ammonia (73.3%), total nitrogen (68.1%) and chemical oxygen demand (COD) (39.3%).They obtained fungal growth on this effluent with an accumulation of biomass (451.2 mg l -1 ) that contained protein (47% g g -1 ).There was variability in fungal capacities as to the removal of pollutants (see Table 1).In fact, Trichothecium roseum was the best in phosphate removal (97.5%), whilst Epicoccum nigrum, Geotrichum candidum and Trichoderma sp. were the best in the removal of ammonia (84%), total nitrogen (86.8%) and COD (72.3%), respectively.Concerning cell-protein production, Paecilomyces carneus had the highest ratio of protein to biomass (92.5%).However, this fungus did not grow very well on domestic sewage.In our laboratory, domestic wastewater pretreatment by a strain of A. niger has been investigated under transient conditions.This fungal biomass removed about 72% of COD and 65% of protein (Coulibaly, 2002).Despite the differences between the bioprocess investigated in these two studies, COD and protein removal rates are in the same order.The overall feasibility of domestic wastewater treatment under sewer-simulating conditions has been explored recently both experimentally and by simulation (Coulibaly, 2002;Coulibaly et al., 2002;Coulibaly and Agathos, 2003).The heat treatment liquor (HTL) of an activated sludge was decolourised by Coriolus hirsutus (Fujita et al., 2000).This fungal strain exhibited a strong ability to decolourise HTL (70%) with an accumulation of manganese independent peroxidase (MIP) and manganese peroxidase (MnP).Optimising the culture medium by adding nitrogen and carbon sources and improving the biomass quality resulted in increased colour removal capacity by C. hirsutus (Kumar et al., 1998;Miyata et al., 2000;Fujita et al., 2000).Although fungal applications have shown good capacities on sewage treatment, they are still underutilised in practice.This could be explained, in part, by a widespread a priori assumption that fungal strains do not perform as well as bacteria.
The influence of co-substrate (see Table 3) upon paper and pulp industrial wastewater treatment, detoxification and decolourisation rates has also been observed with Ceriporiopsis subvermispora, P. chrysosporium, Trametes versicolor, Rhizopus oryzae and Rhizomucor pusillus (Manzanares et al., 1995;van Driessel and Christov, 2002;Nagarathnamma and Bajpai, 1999;Nagarathnamma et al., 1999).The mechanisms of decolourisation of agroindustrial effluents by fungi are reported to include biosorption and/or biodegradation (Ohmomo, 1988;Sayadi and Ellouz, 1995;Soares and Duran, 1998;Christov et al., 1999;Nagarathnamma et al., 1999).Some "mycoreactors" such as rotating biological contactor (MYCOR), trickling filter reactor (MYCOPOR) and continuous column reactor have been developed to decolourise pulp and paper wastewaters (Eaton et al., 1982;Messner et al., 1990;Bajpai et al., 1993).These reactors were able to run over several weeks by maintaining their colour removal rates.Ligninolytic enzymes are also involved in the degradation of organic compounds, including dyes (see below), within these effluents (Chivukula et al., 1995).The enzymatic oxidation mechanism of those pollutants has been well discussed elsewhere and is not the aim of this contribution (Young and Yu, 1997;Mester and Tien,

Treatment Effluents Fungi
Reactor and medium handling Parameters

DYED EFFLUENTS
The effluents of pharmaceutical industries, dyeing, printing, photographs, textile and cosmetics contain dyes (McMullan et al, 2001).For example, over 7 X 10 7 tons dyes are produced annually worldwide, of which about 10% are lost in industrial effluent (Vaidya and Datye, 1982).Wastewaters from textile industries are a complex mixture of many polluting substances such as organochlorine-based pesticides, heavy metals, pigments and dyes.Their compositions have been discussed in detail by O'Neill et al. (1999).The majority of these dyes are slowly removed by the WWTP, because of their toxicities to indigenous microorganisms.Dye removal from wastewaters by established WWTP processes are expensive and need careful application (Vandevivere et al., 1998;Robinson et al., 2001).Furthermore, following anaerobic digestion, nitrogen-containing dyes are transformed into aromatic amines that are more toxic and mutagenic than the parent molecules (Shaul et al., 1985;Chung and Stevens, 1993;Ganesh et al., 1994).To overcome these difficulties, fungi are being investigated for their potential to decolourise effluents.Among them, the most widely studied are the white-rot fungi P. chrysosporium (a model, primarily laboratory organism) and T. versicolor (a promising organism for industrial applications).

METAL CONTAINING EFFLUENTS
Metallurgical industries, mining, surfaces cleaning, waste incinerators produce large wastewater polluted by metals.Dissolved metals escaping into the environment pose a serious health hazard.Because they accumulate in living tissues throughout the food chain, which has human at its top.There is a need to remove heavy metals before they enter the complex ecosystem.Physicochemical treatments evolved in very diluted water-containing metals (precipitation, electrochemical, flocculation, coagulation, ion exchange) are expensive.Utilization of biomasses in general (Volesky, 1994;Veglio and Beolchini, 1997;Kratochvil and Volesky, 1998;McKay et al., 1999;Gupta et al., 2000) and particularly that of fungi are considered to be best alternatives for those waters purification (Kapoor and Viraraghavan, 1995;Modak and Natarajan, 1995;Sag et al., 1998;Volesky and Holan, 1995;Atkinson et al., 1998;Kratochvil and Volesky, 1998;Mogollon et al., 1998;Savvaidis, 1998;Tobin and Roux, 1998).Indeed, the purification of the watercontaining metals by fungal biomass is cheaper and it presents the following advantages: (i) production of residual small volume; (ii) possibility of valorisation of fungal waste biomasses from industrial fermentations; (iii) fast removal and (iv) easy installation of the process.
To increase fungal biomasses removal capacities, some of them undergo physicochemical treatments (soda or acidic treatments, insertion of functional groupings, heat treatment) (Akthar et al., 1995;Akthar and Mohan, 1995;Kapoor et al., 1999;Kramer and Meisch, 1999;Yin et al., 1999;Yan and Viraraghavan, 2000).Moreover, A. niger biomass treatment by soda, makes it possible to adsorb 2.5 to 1000 mg Ag l -1 of Ag + in polluted water (Akthar et al., 1995).In the same way, Kapoor et al. (1999) obtained with a soda treated biomass of A. niger, the removal rates of Cd 2+ , Cu 2+ and Pb 2+ superiors to that of activated carbon (F-400).Akthar and Mohan (1995) used the same type of biomass like precedent authors, and they obtained the removal rates of Zn 2+ and Cd 2+ superiors to that of Dowex-50 resin.An insertion of carboxyl and amino groups in A. niger biomass walls, makes it possible to obtain an adsorption rate ranging in the order of 172-1064 mmol (kg biomass) -1 for Cd 2+ , Co 2+ , Ni 2+ and Zn 2+ (Kramer and Meisch, 1999).A simple detergent and alkaline solutions treatment of M. rouxii biomass was sufficient to obtain an increase in its adsorption capacity (Yan and Viraraghavan, 2000).Fungal biomasses that have sequestered metals can be regenerated following their washing with HNO 3 (0.05 N) and/or with Ca 2+ , Mg 2+ and K + (0.1 M) (Akthar et al., 1995(Akthar et al., , 1996;;Kapoor et al., 1999).

DISCUSSION
Essential works on fungal utilization for raw wastewaters biopurification have been laboratory tests.This situation can be explained by the fact that fungal utilization in environmental biotechnology is still under investigation to assess information's on process implementation.To gain confidence with the results, these investigations are performed on synthetics wastewaters.The good results obtained in laboratory tests depend on growth medium optimisation (addition of co-substrate, nutrients, mediators molecules, physical parameters optimisation) and a good handling of biomasses.However, these works amongst other things prove some advantages when a mycoreactor is introduced in effluents treatment lines.In fact, there are some reductions of bactericidal effects and an increase in biogas production.Consequently work on pilot and the development of treatment plants are to be encouraged.
The degradation and the mineralisation of some recalcitrant dyes and organochlorinated compounds are effective by certain white rot fungi.However fungal aptitudes for raw wastewaters remain dependent on salts concentration, culture conditions and especially on the amendment of carbon and nutriment sources.Among the co-susbstrates tested for effluent pretreatment by fungi, glucose and sucrose were the best, when they were used at 5 to 10 g l -1 .To minimise the mycoreactor integration cost in the treatment line, the co-substrate could be provided by feeding the reactors with amylaceous effluents or others wastewaters rich in sucrose or glucose as these carbon sources proved to be the best cosubstrate.About the growth medium impact on fungal capacities to decolourise HTL, one could use C. hirsutus in post processing of an activated wastewater, because of its sensitivity to organic nitrogen.The oxidoreductases activities could be more significant, thanks to the use of substrate that could ensure the role of mediators' molecules and guarantee the generation of H 2 Ò 2 in the reaction medium.Salts constraint could be overcome while proceeding to the desalinisation of the effluents before their treatment with fungi.
As regards metal removal, a standardization of adsorption rates unit and the rigorous description of biomass morphology (pellet diameter) will allow a better comparison of fungal capacities and a guide for the best choice of fungi.Biomass grinding to produce small particles and their engineering to increase their capacities to remove specific metals are promising way for fungal biosorption.Utilization of residual biomasses from fermentation is still minimal nowadays (Knapp and Newby, 1999); one needs to encourage such practice, because this constitutes a way of making use of them.

CONCLUSION
This review highlighted the capacities of certain fungi to pretreat raw wastewaters.However, essential works on this subject are still laboratories tests and they are of less industrial scale application.The white rot fungi are suitable for the degradation of a large variety of pollutants and to produce at the same time metabolites of great added values (proteins, enzymes).However, an optimisation of the culture media in carbon sources or nutrients and mediators molecules is very important to obtain a good output of pollutants degradations.With regard to other fungi, those also contribute to effluents purification with proteins and enzymes productions (for example, A. awamory and A. niger).Some residual biomasses from fungal fermentation, have been used to remove metals and dyes from effluents.Ultimately, the fungal biomasses present many assets for biopurification of wastewaters.

Table 1 .
Examples of fungi used to treat domestic sewage, starch processing and metal bearing effluents.Optimal culture condition and the effect of fungal pretreatment are reported.

Table 2 .
Examples of fungi used to treat distillery wastewaters.Optimal culture condition and the effect of fungal pretreatment are reported.

Table 3 .
Examples of fungi used to treat wood processing wastewaters.Optimal culture condition and the effect of fungal pretreatment are reported.