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Fungal growth is difficult to quantify, as fungi do not grow as single cells, but as hyphae that cannot be quantified using the usual enumeration techniques. Fungi differentiate to produce spores, resulting in large increases in viable counts often with little relationship to biomass (Pitt, 1984). Thus, in order to determine the efficacy of lignocellulose degradation, it is important to know the amount of fungal

mycelium in solid substrate and establish more reliable and convenient methods for measuring fungal growth in solid-state fermentation (SSF). A number of methods have been developed for quantifying fungal growth and their principles and applications comprehensively reviewed (Matcham et al., 1984; Hartog and Notermans, 1988; Williams, 1989; Newell, 1992; Samson et al., 1992; de Ruiter et al., 1993; Pitt and Hocking, 1997). The conventional microscopic method has long been used. However, it is known to be easy to create measurement errors (de Ridder- Duine et al., 2006).

Ergosterol is found as the main sterol in fungal cell membrane, as a minor component in many plants and is also found in most yeasts and algae (Pasanen et al., 1999; Nielsen and Madsen, 2000; Hippelein and Rugamer, 2004; de Ridder-Duine, 2006). It is specifically a major component of mycelia, spores, and vegetative cells (Newell, 1992; Pasanen et al., 1999) and plays a role in several biochemical processes such as membrane fluidity, cation permeability and cell growth (Bossche, 1990; Hippelein 2004). Since there is a good relationship between ergosterol content and hyphal length, ergosterol is recommended for quantifying fungal growth (Schnurer, 1993; Pasanen et al., 1999). However, the amounts of ergosterol are known to vary between different fungal species, period of culture, stages of development, and growth conditions (Gessner and Chaucet, 1993; Newell, 1994; Schnurer, 1993; Pasanen et al., 1999). Ergosterol measurement is recognised as a biomarker for assessing fungal biomass in solid media or soil, since it is difficult to measure mycelial biomass directly when closely associated with the substrate (Niemenmaa et al., 2008). Montgomery et al. (2000) suggested to use a conversion factor to calculate fungal biomass from ergosterol concentration.

The average ergosterol content in several fungal species ranges from 2.6 to 42 µg ml-1 of dry mass (Pasanen et al., 1999), while Hippelein and Rugamer (2004) reported that ergosterol concentration in several fungi varies from 0.1 to 130 µg g-1 of dry mass. Moreover, Montgomery et al. (2000) obtained an average of 4 g ergosterol/mg of biomass using six species of fungi, in which per 250 µg dry fungal biomass was the equivalent of 1 µg of ergosterol. In liquid cultures the conversion factors for ergosterol to biomass has been shown to vary from 48 to 243 and 185 to 88 for P. radiata and P. chrysosporium respectively (Niemenmaa et al. 2008). However, this conversion factor cannot be applied to the other type of fungi due to different fungal physiologies and other factors such as media, substrate, and nutrients, along with different fungal growth stages (Klamer and Baath, 2004). Despite the limitation of ergosterol as an indicator of fungal biomass, it has been applied to a wide range of environments such as soil (Gong et al., 2001; Ruzicka et al., 1995: Ruzicka et al., 2000); building material (Hippelein and Rugamer, 2004); indoor environment (Flannigan, 1997); house dust (Saraf et al., 1997) ; grain (Borjesson et al., 1990) ; seeds (Richarson and Logendra, 1997) ; plant litter (Gessner and Newell, 2002) ; plant material (Newell, 1992) agar media, and wood (Niemenmaa et al., 2006). Gong et al. (2001) reported that the ergosterol content of soil samples ranged between 0.34 to 2.5 µg g-1, whilst the amount of ergosterol in fungal agar and wood substrate varied between 149 to 539.5 µg g-1 and 15.3 to 42.9 µg g-1, respectively (Niemenmaa et al., 2006). These results show a diverse range in ergosterol content between fungal types and the culture media. However there is little information available for the white rot, and brown rot fungi (basidiomycetes),

therefore studies of ergosterol fungal biomass grown in wheat straw SSF are needed to determine the efficacy of this assay for these species.

Several methods of ergosterol extraction are used: (1) free ergosterol which is soluble in alcohol, (2) total neutral ergosterol from saponification of alcoholic extract, (3) total alkali ergosterol which is extracted and saponified in alkaline alcohol solution (Davis and Lamar, 1992; Hippelein and Rugamer, 2004). Gong et al. (2001) developed a physical disruption technique to simplify extraction by replacing the time-consuming reflux step. In order to maximize ergosterol release from fungal membranes, Ruzicka et al. (1995) proposed using an ultrasonic method in combination with non-alkaline extraction for ergosterol sample from clay loam soil. Following extraction ergosterol abundance was measured using normal phase HPLC. This method has been limited to measuring the ergosterol from soil, and has not previously been used to measure fungal growth during SSF. We used this method to estimate fungal biomass of different types of brown rot and white rot fungi (Basidiomycetes). It has been reported that measuring ergosterol is more accurate than other fungal biomass estimation approaches that measure molecules such as chitin or adenosine triphospate (ATP) (Klamer and Baath, 2004). However, another method for measuring fungal biomass such as phospolipid fatty acid (PLFA) has also been used in combination to combine with the ergosterol method. PLFA has been used as another fungal profiling parameter for minimizing the uncertainty of ergosterol assay results and as a complement to measurement of fungal biomass (de Ridder-Duine, 2006; Klamer and Baath, 2004). Total PLFA content has been found to have a positive correlation with bacterial or fungal biomass, at the same time it can be distinguish the fingerprint of microbial communities (Frostegard and Baath,

1996). The PLFA 18:2n6, represents PLFA for fungi (Klamer and Baath, 2004), since it represent 45% of fungal body (Federle, 1986) and is absent in bacteria. This marker is also used for fresh plant biomass (Frostegard and Baath 1996; Snajdr et al., 2011), however the content of this PLFA (18:2n6) in dead plant biomass decreases rapidly (Snajdr et al., 2011).

The PLFA assay has been used successfully to monitor P. chrysosporium biomass during its cultivation on rice straw (Yu et al., 2009). A relationship between PLFA (18:2ω6,9) and ergosterol content gave a conversion factor of 1.47 µmol 18:2ω6,9 to 1 mg ergosterol using 12 different fungal species in PDA culture (Klamer and Baath, 2004). In pure culture, Eiland et al. (2001) recorded a value of 2.1 µmol 18:2ω6,9 to 1 mg ergosterol. PLFA (18:2ω6,9) depending on growth and the type of ecosystem (e.g soil and compost). Only a few attempts have been made at determining fungal biomass from direct PLFA measurement (Klamer and Baath, 2004). PLFA does not provide a good taxonomic marker (Lechevalier and Lechevalier (1988) as there is a possibility of obtaining similar fatty acid fingerprints for both Ascomycetes and Basidiomycetes. Multivariate analysis is a powerful tool for distinguishing fatty acid profiles and profiling using this approach for fungi was proposed. Muller et al., (1994) studied the differences in profiles of fatty acid, fatty acids and sterols of fungi using multivariate discriminant analysis.

The objective of the present study was to monitor sequential changes in ergosterol and PLFA 18:2ω6,9 content from selected Basidiomycetes cultured in SSF, in order to estimate fungal biomass. A multivariate statistical analysis was also employed to distinguish the PLFA fingerprint for each fungal species.