Appl Microbiol Biotechnol (2007) 74:547554 DOI 10.

1007/s00253-006-0695-9

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Growth and laccase production kinetics of Trametes versicolor in a stirred tank reactor
A. T. Thiruchelvam & Juliana A. Ramsay

Received: 30 June 2006 / Revised: 3 October 2006 / Accepted: 9 October 2006 / Published online: 11 January 2007 # Springer-Verlag 2007

Abstract White rot fungi are a promising option to treat recalcitrant organic molecules, such as lignin, polycyclic aromatic hydrocarbons, and textile dyes, because of the lignin-modifying enzymes (LMEs) they secrete. Because knowledge of the kinetic parameters is important to better design and operate bioreactors to cultivate these fungi for degradation and/or to produce LME(s), these parameters were determined using Trametes versicolor ATCC 20869 (ATCC, American Type Culture Collection) in a magnetic stir bar reactor. A complete set of kinetic data has not been previously published for this culture. Higher than previously reported growth rates with high laccase production of up to 1,385 U l1 occurred during growth without NH 4 or glucose limitation. The maximum specific growth rate averaged 0.940.23 day1, whereas the maximum specific substrate consumption rates for glucose and ammonium were 3.371.16 and 0.150.04 day1, respectively. The maximum specific oxygen consumption rate was 1.63 0.36 day1. Keywords Growth . Kinetics . Laccase . Stirred tank reactor . Trametes versicolor

Introduction White rot fungi (WRF) have been used successfully to treat hazardous and recalcitrant organic molecules such as polycyclic aromatic hydrocarbons (Field et al. 1992; DAnnibale et al. 2005), phenols (Ryan et al. 2005),
A. T. Thiruchelvam : J. A. Ramsay (*) Department of Chemical Engineering, Queens University, Kingston, ON K7L 3N6, Canada e-mail: juliana.ramsay@chee.queensu.ca

polychlorinated biphenyls (Krcmar et al. 1999), and textile dyes (Knapp et al. 1995; Heinfling et al. 1997; Swamy and Ramsay 1999; Borchert and Libra 2001), effluents from pulp plants (Livernoche et al. 1983; Chang et al. 1987; Prasad and Joyce 1991; Bajpai et al. 1993; Font et al. 2003), sugar refineries (Guimaraes et al. 2005), and pulp bleaching (Addleman and Archibald 1993), respectively. WRF produce extracellular, lignin-modifying enzymes (LMEs) that play a key role in such processes. These highly nonspecific enzymes include laccase, manganese peroxidase, and lignin peroxidase. Although there is increasing interest in using these fungal cultures or the enzymes that they produce, there is little published data on the growth or enzyme production kinetics of WRF (Table 1). The maximum specific growth rate ( max ) of some WRF such as Phanerochetae chrysosphorium (Barclay et al. 1993), Trametes sp. (Jang et al. 2002), Trametes pubescens (Ryan et al. 2005), and T. versicolor (Tavares et al. 2005) has been reported (Table 1), but other useful values such as the specific rates of substrate (e.g., carbon or nitrogen source) and oxygen utilization or of product (e.g., CO2 or LMEs) formation are seldom found. These kinetic parameters are important to better design and operate a bioreactor (Gaden 2000) whether using WRF for biodegradation or producing LMEs. In our studies, we used T. versicolor ATCC 20869. Kinetic data have not been previously published for this strain, although max and the yield of biomass produced from the glucose substrate consumed (YX/S) for an unknown strain of T. versicolor (Tavares et al. 2005) and some parameters for other species of Trametes are available in the literature (Table 1). Therefore, the objective of this study was to characterize the growth of T. versicolor ATCC 20869 to obtain the yield coefficients, the maximum specific growth rate (max), the maximum

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Appl Microbiol Biotechnol (2007) 74:547554

Table 1 Comparison of the kinetic parameters determined in this study for T. versicolor ATCC 20869 with literature values for other fungi Culture Substrate Reactor Temperature max (C) (day1) qglucose, max (g g1 day1) b 2.554.19

qO2; max (g g1 day1)

a YX =S

qlaccase, max (U g1 day1) 124400

References

White rot fungi P. chrysosporium Trametes sp. T. pubescens T. versicolorc T. versicolor ATCC 20869 Other fungi Pencillium chrysogenum A. niger

Glycerol Glucose Glucose Glucose Glucose

Static flask Shake flask Airlift reactor Shake flask MSBR

37 28 28 28 222

0.49 0.472 0.25 0.82 0.87 0.76 1.24 2.21 2.21 12.113

1.04 1.392.05

0.44 0.17 0.38 0.30 0.37 0.45 0.45 0.12 0.22

Barclay et al. (1993) Jang et al. (2002) Ryan et al. (2005) Tavares et al. (2005) This study

Glucose Glucose Cane molasses

Stirred tank

25 25 30

Birol et al. (2002) Shuler and Kargi (1992) Ali et al. (2002)

g of biomass g1 of carbon substrate Not reported c Strain not reported

a b

specific consumption   rates of glucose (qglucose, max), ammonium qNH q ; and O O 2 ; max 2; max and the specific 4 production rates of CO2 qCO2; max and laccase (qlaccase, max) in a stirred tank reactor. The yield coefficients  for biomass  produced from glucose ( Y ), ammonium YX=NH ; and X/S 4 O2 YX=O2 consumed were determined.

Materials and methods Inoculum preparation T. versicolor ATCC 20869 grown on malt agar plates and stored at 4C was used to inoculate autoclaved (at 121C for 20 min) 500-ml Erlenmeyer flasks containing 200 ml of modified Kirks medium (Shin et al. 2002) with 2 g l1 ammonium tartrate. The contents of the flasks were incubated for 5 to 7 days at 222C and 180 rpm on a New Brunswick Scientific shaker (Innova 2000). Small pellets (<1-mm diameter) were recovered by decantation after the larger pellets had settled by gravity. The contents of the two flasks were combined and further concentrated to a final volume of about 100 ml. The modified Kirks medium was designed to be neither glucose nor nitrogen limited. Bioreactors Two reactors, a BioFloIIC fermentor (New Brunswick Scientific) with three Rushton impellers and a magnetic stir bar reactor (MSBR), were used for the batch fermentation experiments.

BioFloIIC batch fermentation The BioFloIIC fermentor was equipped with a sparger ring at the base of the impellers, a sampling line (10-mm diameter), a polarographic dissolved O2 (DO) probe (Ingold Inpro 6000 series), and pH control (Ingold model 465 probe). The fermentor was filled with 1.25 l of modified Kirks medium and autoclaved for 30 min at 121C. After adding 100 ml of inoculum, 25-ml samples were taken. Fermentations were performed at 222C and at pH 4.55.5. Impeller speeds of 200350 rpm and airflow rates of 0.2 1 vvm were adjusted manually to maintain a minimum of 60% saturation of DO. An infrared CO2 analyzer (Qubit S152, 03%) measured CO2 in the exhaust gas. MSBR batch fermentation The MSBR (Fig. 1) consisted of a 5-l glass bottle (150 mm in diameter200 mm in height) with a magnetic stir bar (12.7-mm diameter76.2 mm length) and a magnetic stirrer (ColePalmer model 4815). The bottle was sealed with a rubber stopper with two 5-mm-diameter stainless steel tubes connected to 0.45-m filters to serve as air inlet and outlet. The MSBR was filled with 1.25 l of Kirks modified medium and autoclaved for 30 min at 121C. Inoculation and sampling procedures were the same as with BioFloIIC. The stirring speed was adjusted such that the bottom of the vortex in the culture touched the magnetic stir bar. A buffer, 2,2-dimethylsuccinate, was used to maintain the pH at about 4.5.

Appl Microbiol Biotechnol (2007) 74:547554 Fig. 1 Schematic diagram of MSBR experimental setup

549

Headspace gas was continuously withdrawn at 0.3 l min1 and analyzed for O2 (Qubit S102) and CO2 (Qubit S152, 03%). At certain sampling times, the system was closed by connecting the outlet to the inlet of the headspace such that the headspace gas was recirculated, and the O2 and/or CO2 data with time (Fig. 2) were acquired into a computer to determine qO2 and qCO2 : An average respiratory quotient (RQ=0.820.09) was determined in the MSBR-2 fermentation. In all other fermentations, CO2 was measured, and O2 consumption was determined from the CO2 produced and from the experimentally determined RQ.

Overall oxygen transfer coefficient of the MSBR The unsteady state gassing-out method (Shuler and Kargi 1992) was used to determine the O2 transfer coefficient (KLa) of the MSBR at 222C. The DO probe was calibrated at 0 and 100% saturation after sparging with N2 and air, respectively, for 30 min. To measure KLa, 1- to 1.35-l distilled water was sparged with compressed air, then sparged with N2 to 10% saturation of DO. The headspace was quickly flushed with air, and the increase in DO of the water was measured with time (Fig. 3) under the same conditions as the fermentations. Because there was no O2

100
20.8 2.8 2.4 20.0 19.6 19.2 18.8 18.4 18.0 0 0.4 0.8 1.2 1.6 2 2.4 2.0 1.6 1.2 0.8 0.4 0.0

0.5 A 0.0 -0.5 ln [(c* - c)/(c* - co )]

80 DO (%) 60

20.4

CO 2 in headspace (%)

O2 in headspace (%)

-1.0 40 B 20 0 0 2 4 6 8 10 12 14 Time (min) -1.5 -2.0 -2.5

Time (h)
Fig. 2 A typical set of data for O2 consumption and CO2 production rates determined in a closed MSBR for MSBR-2 fermentation

Fig. 3 A typical set of results from abiotic KLa determination using the degassing method. N2 sparging was started at A and stopped at B when the headspace was also quickly flushed with air. The DO data between the dashed lines were linearized for KLa determination according to Eq.(1)

550

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consumption, the KLa (day1) was calculated according to the equation:   c c KL at ln 1 c co where c*=saturation concentration of O2 in water (mg l1), c=concentration of O2 in water at t=t (mg l1), and c0=concentration of O2 in water at t =0 (mg l1). Biomass determination The biomass from each sample was retained on 1.2-m Millipore filters, washed with about 150 ml of distilled water, and then dried at 103C for 24 h before weighing. For verification purposes, a single triplicate biomass analysis from a MSBR run was averaged from three 25-ml samples and was found to be 2.93 0.28 g l1. The supernatant was analyzed for laccase activity, and then frozen for subsequent assays. Laccase assay Laccase activity was determined by measuring the rate of color generation due to the enzymatic oxidation of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS radical ion ABTS at 420 nm and pH 5.0. The extinction coefficient () used was 36,000 M1 cm1 (Wolfenden and Willson 1982). The assay solution contained 100-mM sodium acetate as the buffer, 0.2 mM ABTS, and an appropriate amount of sample. Production of 1 M ABTS per min was defined as one unit of activity (U l1) of laccase. Glucose determination Glucose was determined by a colorimetric method (Lever 1972). The assay reagent was prepared by mixing 5% w/v solution of p-hydroxybenzoic

acid hydrazide in 0.5 M HCl with 0.5 M NaOH at a ratio of 1:9 by volume. Fifteen microliters of sample was added to 4.5 ml of the reagent and heated in boiling water for about 6 min. After cooling in a cold-water bath for 3 min, the absorbance was measured at 410 nm. Using a calibration curve, the concentration of glucose was determined.
NH 4 determination NH 4 was determined by the phenolhypochlorite method (Weatherburn 1967). A 40-l sample was mixed with 2.5 ml of reagent-1 (10 g of phenol and 50 mg of sodium nitroprusside in 1 l of distilled water) and 2.5 ml of reagent-2 (5 g of NaOH and 8.4 ml of sodium hypochlorite in 1 l of distilled water), and left for 30 min at room temperature. The reaction was slowed in a cold-water bath for 5 min, and the absorbance was measured at 630 nm within 30 min. The NH 4 concentration was determined from a calibration curve. Glucose, NH 4 ; and laccase assays were done in duplicate and the average reported.

Results BiofloIIC fermentations In the BioFloIIC fermentor, the fungus formed pellets of 0.5 to 3 mm in diameter. As batch fermentations progressed, the biomass attached to the impellers, probes, and reactor surfaces, especially around the inlet/outlet ports, when the reactor liquid volume decreased to their levels due to sampling. This caused scatter in the biomass data. Glucose and ammonium data were smoother. Some growth parameters were calculated

Table 2 Summary of the batch kinetic data of T. versicolor in suspended culture in a stirred tank reactor (BioFloIIC) with Rushton impellers or a MSBR at 222C in Kirks medium Parameter BioFloIIC 1 Initial glucose (g l1) Initial ammonium (g l1) Inoculum size (g l1) max (day1) qglucose, max (g g1 day1) 1 1 qNH ; max (g g day ) 4 qO2; max (g g1 day1) qCO2; max (g g1 day1) 10 0.40 0.45 0.56 4.40 0.15 nd nd nd 0.19 6.9 2 33 1.30 0.06 0.88 nd nd nd nd nd 0.34 8.0 MSBR 1a 10.5 0.33 0.01 0.40 1.17 nd nd nd nd 0.36 nd 2 11.0 0.33 0.15 1.24 4.19 0.13 2.05 2.31 400 0.30 11.0 3b 9.5 0.4 0.14 0.76 2.55 0.19 1.39 1.69 124 0.37 8.4 4 10 0.31 0.1 0.77 nd nd 1.46 1.65 315 nd nd 5 10 0.36 0.1 1.00 nd 0.12 nd nd 399 nd 9.1

qlaccase, max (U g1 day1) YX/S (g biomass g1 glucose) YX=NH (g biomass g1 ammonium) 4

Values determined from graphs like Fig. 5 nd Not determined a Low inoculum size b Data from fermentation in Fig. 4

Appl Microbiol Biotechnol (2007) 74:547554

551

(Table 2) from two fermentations, where the scatter in biomass data was minimal. MSBR fermentations In the MSBR, T. versicolor formed pellets of uniform size in the range of 1 mm. The pellets were homogeneously distributed with little biomass attachment, giving more meaningful results than the data obtained from the BioFloIIC, leading to more confidence in the kinetic analyses. The configuration of the stir bar may have been an important factor. It had two O-rings (2mm thick) at the center and one at either end. Without the O-rings, the fungal pellets were much smaller than 1 mm, and lower laccase production was obtained (100200 U l1 compared to about 900 U l1). Oxygen transfer in MSBR Using DO vs time data (Fig. 3) and Eq. (1), KLa values of the MSBR with distilled water were determined to be 225 days1 and 720 days1 for 1.35 l (i.e., start-up volume of the MSBR) and 1.0 l (i.e., final reactor volume), respectively, under operating conditions. Growth kinetics Five batch fermentations were performed in the MSBR with a typical set of results shown in Fig. 4. Using polynomial curves, slopes were calculated then divided by the biomass concentration at the corresponding time to determine the specific rates (e.g., Fig. 5) from which the maximum specific rates were identified (Table 2). Except for MSBR-1 where max was low (0.40 day1), probably because of a low inoculum size (0.01 g l1), the MSBR max values were generally higher than those of the BioFloIIC and ranged from 0.76 to 1.24 day1. There was a similar variation in the biomass yield from NH 4 (8.411) and from glucose (0.300.37; Table 2).

3.0 Specific rates (day ) 2.5 2.0 1.5 1.0 0.5 0.0 3.0 Specific rates (day ) 2.5 2.0 1.5 1.0 0.5 0.0 0
-1

a)

qglu qamm (x 0.1) qO2

-1

b)

qlac qCO2

Time (day)
Fig. 5 Specific rates calculated from the data shown in Fig. 4

10 8 Glucose (g/l) + Lac (x 100 U/l) 6 4 2 0 0 2 4 6 8 10 Time (day)

5 Biomass (g/l) Ammonium (x 0.1 g/l) 4 3 2 1 0

The O2 uptake and CO2 production rates of MSBR-2 were calculated from the headspace O2 and CO2 data as in Fig. 2. Because the CO2 analyzer was more sensitive than the O2 analyzer, specific O2 uptake rate qO2 was routinely determined using the CO2 data and the average respiratory quotient (RQ=0.820.09) from the MSBR-2 fermentation. The average qCO2 ;max and qO2 ;max values were 1.880.37 g (g biomass)1 day1 and 1.630.36 g (g biomass)1 day1, respectively (Table 2). Laccase production kinetics The relationship between growth and laccase production was examined from plots of and qlaccase as a function of time (e.g., Fig. 5) for four MSBR fermentations. The difference in time when the maximum specific growth rate tmmax and laccase production rate (tqlaccase,max) were achieved is defined as t tqlaccase; max tmmax and was used to determine the degree to which laccase production was growth associated. Laccase production was partially growth associated. The t was 0.530.21 day when max ranged from 0.76 to 1.24 day1 (Table 3).

Fig. 4 Typical batch data from MSBR experiment where little biomass attachment problem was encountered. All glucose, and laccase assays were done in duplicate

552 Table 3 Comparison of growth and laccase production rates in the MSBR experiments MSBR Growth max (day1) 2 5 4 3 1 1.24 1.00 0.77 0.76 0.40 Xmax (g l1) 2.5 3.1 3.0 2.7 2.9 Laccase production

Appl Microbiol Biotechnol (2007) 74:547554

t (day) laccasemax (U l1) 864 1,385 920 688 1,515

max)

tmmax (day)
2.0 3.0 2.8 2.5 na

qlaccase, 400 399 315 124 na

max

(U g1 day1)

tqlaccase, 2.3 3.5 3.4 3.0 na

max

(day) 0.3 0.5 0.8 0.5 na

t is used as an index of growth associatedness, where t=time at which qlaccase, max is achieved (tqlaccase, achieved tmmax : As t increases, laccase production is increasingly less growth associated. na Not applicable a Inoculum size was low; was constant with increasing qlaccase.

minus the time at which max is

Discussion Growth habit Because significant biomass attachment was encountered in the batch BioFloIIC fermentation, the data from these experiments were not as reliable as those obtained from the MSBR where little biomass attachment was seen. Attachment, especially towards the end of fermentation when ammonium levels decreased to less than 0.1 mg l1, could be due to extracellular polysaccharide that may make the fungal biomass more adherent. Several strains of basidiomycetes, including a T. versicolor strain, produced more polysaccharide towards the end of the fermentation (Maziero et al. 1999) and Moreira et al. (1998) reported that, under nitrogen limitation, pellets of Phanerochaete chrysosporium tend to stick together due to extracellular polysaccharide. T. versicolor, a filamentous fungus, grows by extending its hyphal tips unlike bacteria that reproduce by binary fission. When grown in shake flasks on a gyrorotary shaker (150200 rpm) or in the BioFloIIC reactor under our experimental conditions, it formed pellets. In the latter, the pellet size varied from about 0.5 to 3 mm, depending on the type of mixing, and increased in size as the fermentation progressed. Smaller pellets (1 mm) were always formed in the MSBR compared to BioFloIIC fermentor. Because not all cells in the fungal mycelium are involved in growth, the growth rate will not depend on the amount of biomass but on the number of growing tips. However, if the mycelia are broken, the number of growing tips will increase, and growth may be more closely related to the quantity of biomass (Cui et al. 1998). This is more likely the case in the MSBR where smaller pellets and a more homogeneous suspension were observed. Oxygen transfer The oxygen transfer rate in the MSBR and in the BioFloIIC were similar. In this study, the measured abiotic KLa in the MSBR ranged from 225 to 720 days1 as the working volume decreased (1.35 to 1.0 l). Gupta and

Rao (2003) reported KLa values of 500800 days1 for distilled water at 37C and impeller speeds of 200350 rpm in a BioFloIII (essentially the same bioreactor). Due to their good oxygen transfer capabilities, continuous stirred tank reactors with Rushston impellers, such as the BiofloIIC, are preferred for the examination of microbial kinetics. However, the KLa of the MSBR was found to be similar to that of the Bioflo, although it did vary considerably due to the effects of the vortex as the reactor volume decreased. Thus, pellet diameter rather than KLa would be the main factor leading to O2-limited growth kinetics. Wittler et al. (1986) and Cui et al. (1998) have shown that fungal pellet diameters up to 300 m may not be O2-limited depending on the species and growth conditions. The MSBR allowed bead diameters to be maintained less than 1 mm in diameter, thus minimizing the effects of O2 limitation. Growth kinetics The average max of T. versicolor was 0.940.23 day1 in batch MSBR cultures. Growth was much slower when compared to deuteromycetes such as Aspergillus niger that can have a max as high as 12.96 days1 (Ali et al. 2002). However, the max values in the MSBR fermentor were higher or the same order of magnitude as those obtained for other Trametes (Table 1). Using a similar modified Kirks medium composition with 20 g l1 glucose and 4 g l1 ammonium tartrate at 28C, Jang et al. (2002) obtained a lower max of 0.472 day1 but a much higher YX/S of 0.87 with Trametes sp. when compared to our study (YX/S of 0.340.038), where T. versicolor was grown at a lower temperature (22C; Table 2). Thus, although T. versicolor grew more quickly in our study, a significant portion of the carbon consumed (about 66%) was not converted to biomass. However, in shake flask experiments at 28C, Tavares et al. (2005) obtained similar max values (0.82 and 0.87 day1, with and without glucose limitation, respectively) and YX/S (0.17 and 0.38, with and without glucose limitation, respectively) for an unknown strain of T. versicolor grown in a Trametes-defined medium.

Appl Microbiol Biotechnol (2007) 74:547554

553

When Ryan et al. (2005) cultivated T. pubescens CBS 696.94 on a complex medium containing glucose (10 g l1) and peptone (10 g l1) in an airlift reactor at 28C, a lower max of 0.25 day1 was obtained. Although grown at a lower temperature than Jang et al. (2002), Tavares et al. (2005), and Ryan et al. (2005), this is the highest reported max for a Trametes culture and may be a consequence of the particular characteristics of the MSBR. The pellet size was smaller in the MSBR than in the BiofloIIC or shake flasks probably due to higher shear, where the magnetic stir bar was in contact with the reactor base. Although high shear stress has been shown to cause cell damage (Hess et al. 2002), the max of T. versicolor in the MSBR may have been enhanced by the reduction in mass transfer limitation due to a smaller pellet size. The average qO2 ; max of our T. versicolor was 1.63 0.36 day1 in the MSBR. Although T. pubescens had a much lower max (0.25 day1), its qO2 ; max of 1.04 day1 (Ryan et al. 2005) was high in terms of yield of biomass from oxygen, YX=O2 : For T. pubescens, YX=O2 was 0.24 compared to 0.560.04 g biomass (g O2)1 determined for T. versicolor in this study. This difference could be due to the reduced mass transfer limitations in the smaller pellets in the MSBR. Pellet size in Ryan et al. (2005) was estimated to be 34 mm compared to 1 mm in this study. Maximum specific substrate consumption rates are also reported in this study for the first time for T. versicolor in a stirred tank reactor, and the average values for glucose and 1 NH and 0.15 4 were found to be 3.371.16 day 1 0.04 day , respectively. Laccase production kinetics High laccase production can be achieved by adding inducers (Tavares et al. 2005; Galhaup et al. 2002) such as Cu2+, phenol, or 2,5-xylidine at a certain stage in the growth phase. In the MSBR studies, the fermentations were performed without inducers. However, the qlaccase from our experiments (310130 U g1 day1) was much higher than the value (63 U g1 day1) reported by Tavares et al. (2005) under nonglucose-limited growth. This may be due to the difference in the reactor type and strain because Tavares et al. (2005) carried out the experiments in shake flasks with an unidentified strain of T. versicolor. Previous studies have shown that production of LMEs, such as laccase, is usually secreted as secondary metabolites when a nutrient such as glucose or ammonium is limiting (Kirk and Farrell 1987; de Jong et al. 1994; Tavares et al. 2005). In our studies, without any inducers added, laccase production was more closely associated with growth and was not linked to glucose or ammonium limitation. Again, our results may differ from previous findings because of the mixing characteristics of the MSBR. The shear stress that resulted in smaller pellet size may stimulate laccase production during growth. The

MSBR allowed for the production of Trametes pellets that had high laccase productivities. At a larger scale, similar pellets might be produced with an airlift design incorporating a device in the down-comer that could produce shear effects similar to that of the MSBR. In conclusion, this is the first comprehensive evaluation of the growth and laccase production kinetics by T. versicolor. The kinetic parameters determined in this study can serve as a basis for bioreactor design and process control. Fungal growth in the MSBR had less biofouling problems and produced smaller sized biomass pellets that were more uniformly suspended than what was obtained in a BioFloIIC equipped with Rushton impellers. T. versicolor (max = 0.92 day1) grew more slowly than other fungi. The qs, max for glucose, NH 4 ; and O2 were reported for the first time and were 3.371.16 day1, 0.150.04 day1, and 1.63 0.36 day1, respectively. The yield coefficients for biomass from glucose, NH 4 ; and O2 were 0.340.05, 9.501.35, and 0.560.04, respectively. Laccase production was somewhat growth associated and was not stimulated by nutrient limitation in this reactor.

References
Addleman K, Archibald F (1993) Kraft pulp bleaching and delignification by dikaryons and monokaryons of Trametes versicolor. Appl Microbiol Biotechnol 59:266273 Ali S, Ikram-ul-Haq, Qadeer MA, Iqbal J (2002) Novel technique for microbial production of 3,4-dihydroxy phenyl L-alanine by a mutant strain of Aspergillus oryzae. Electron J Biotechnol 5: 258271 Bajpai P, Mehna A, Bajpai PK (1993) Decolorization of kraft bleach plant effluent with the white rot fungus Trametes versicolor. Process Biochem 28:377384 Barclay CD, Legge RL, Farquhar GF (1993) Modeling the growth kinetics of Phanerochaete chrysosporium in submerged static culture. Appl Environ Microbiol 59:18871892 Birol G, Undey C, Cinar A (2002) A modular simulation package for fed-batch fermentation: penicillin production. Comput Chem Eng 26:15531565 Borchert M, Libra JA (2001) Decolorization of reactive dyes by the white rot fungus Trametes versicolor in sequencing batch reactors. Biotechnol Bioeng 75:313321 Chang H-M, Joyce TW, Kirk TK (1987) Process of treating effluent from a pulp or papermaking operation by white-rot fungi. US Patent 4,655,926 Cui YQ, Okkerse WJ, van der Lans RGJM, Luyben KCAM (1998) Modeling and measurements of fungal growth and morphology in submerged fermentations. Biotechnol Bioeng 60:216229 DAnnibale A, Ricci M, Leonardi V, Quaratino D, Mincione E, Petruccioli M (2005) Degradation of aromatic hydrocarbons by white-rot fungi in a historically contaminated soil. Biotechnol Bioeng 90:723731 de Jong E, Field J, de Bont J (1994) Aryl alcohols in the physiology of ligninolytic fungi. FEMS Microbiol Rev 13:153188 Field JA, de Jong E, Costa GF, de Bont JAM (1992) Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi. Appl Environ Microbiol 58:22192226

554 Font X, Caminal G, Gabarrell X, Romero S, Vicent MT (2003) Black liquor detoxification by laccase of Trametes versicolor pellets. J Chem Technol Biotechnol 78:548554 Gaden EL Jr (2000) Fermentation process kinetics. Biotechnol Bioeng 67:413419 Galhaup C, Wagner H, Hinterstoisser B, Haltrich D (2002) Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 30:529536 Guimaraes C, Porto P, Oliveira R, Motab M (2005) Continuous decolourization of a sugar refinery wastewater in a modified rotating biological contactor with Phanerochaete chrysosporium immobilized on polyurethane foam disks. Process Biochem 40:535540 Gupta A, Rao G (2003) A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor. Biotechnol Bioeng 84: 351358 Heinfling A, Bergbauer M, Szewzyk U (1997) Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta. Appl Microbiol Biotechnol 48:261266 Hess J, Leitner C, Galhaup C, Kulbe KD, Hinterstoisser B, Steinwender M, Haltrich D (2002) Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor. Appl Biochem Biotechnol 98:229242 Jang MY, Ryu WR, Cho MH (2002) Laccase production from repeated batch cultures using free mycelia of Trametes sp. Enzyme Microb Technol 30:741746 Kirk T, Farrell R (1987) Enzymatic combustion: the microbial degradation of lignin. Annu Rev Microbiol 41:465505 Knapp JS, Newby PS, Reece LP (1995) Decolorization of dyes by wood-rotting basidiomycete fungi. Enzyme Microb Technol 17:664668 Krcmar P, Kubatova A, Votruba J, Erbanova P, Novotny C, Sasek V (1999) Degradation of polychlorinated biphenyls by extracellular enzymes of Phanerochaete chrysosporium produced in a perforated plate bioreactor. World J Microbiol Biotechnol 15:269276 Lever MA (1972) New reaction for colorimetric determination of carbohydrates. Anal Biochem 47:273279

Appl Microbiol Biotechnol (2007) 74:547554 Livernoche D, Jurasek L, Desrochers M, Dorica J (1983) Removal of color from kraft mill wastewaters with cultures of white-rot fungi and with immobilized mycelium of Coriolus versicolor. Biotechnol Bioeng 25:20552065 Maziero R, Cavazzoni V, Bononi VLR (1999) Screening of basidiomycetes for the production of exopolysaccharide and biomass in submerged culture. Rev Microbiol 30:7784 Moreira MT, Palma C, Feijoo G, Lema JM (1998) Strategies for the continuous production of ligninolytic enzymes in fixed and fluidised bed bioreactors. J Biotechnol 66:2739 Prasad DY, Joyce TW (1991) Color removal from kraft bleach-plant effluents by Trichoderma sp. Tappi J 74:165169 Ryan DR, Leukes WD, Burton SG (2005) Fungal bioremediation of phenolic wastewaters in an airlift reactor. Biotechnol Prog 21:10681074 Shin M, Nguyen T, Ramsay J (2002) Evaluation of support materials for the surface immobilization and decoloration of amaranth by Trametes versicolor. Appl Microbiol Biotechnol 60:218223 Shuler ML, Kargi F (1992) Bioprocess engineering: basic concepts. Prentice-Hall PTR, Englewood Cliffs, NJ, pp 160, 278279 Swamy J, Ramsay JA (1999) The evaluation of white rot fungi for the decoloration of textile dyes. Enzyme Microb Technol 24: 130137 Tavares APM, Coelho MAZ, Coutinho JAP, Xavier AMRB (2005) Laccase improvement in submerged cultivation: induced production and kinetic modeling. J Chem Technol Biotechnol 80: 669676 Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971974 Wittler R, Baumgarti H, Lubbers DW, Schugerl K (1986) Investigations of oxygen transfer into Penicillium chrysogenum pellets by microprobe measurements. Biotechnol Bioeng 28: 10241036 Wolfenden B, Willson R (1982) Radical-cations as reference chromogens in kinetic studies of one-electron transfer reactions. J Chem Soc Perkin Trans 2:805812