Enzyme and Microbial Technology 46 (2010) 603–609
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Nutrient manipulation as a basis for enzyme production in a gradostat bioreactor S. Govender a,b,c,∗ , V.L. Pillay c , B. Odhav c a
Institut für Biochemie, Technische Universität Dresden, D-01062 Dresden, Germany Council for Scientiﬁc and Industrial Research, CSIR Built Environment, PO Box 395, Pretoria 0001, South Africa c Faculty of Engineering, Science and the Built Environment, Durban University of Technology, Durban 4001, South Africa b
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 17 March 2010 Accepted 18 March 2010 Keywords: Phanerochaete chrysosporium Membrane gradostat bioreactor Manganese peroxidase Nutrient additives Enzyme production
a b s t r a c t Nutrient gradients and a supplemented production medium were manipulated using a membrane gradostat bioreactor for the continuous production of manganese peroxidase (MnP) from immobilised Phanerochaete chrysosporium. A production medium containing Tween 80, soybean asolectin and lecithin was developed and tested on batch vessels and evaluated on a continuous single capillary membrane gradostat reactor (SCMGR). The enzyme productivity proﬁle in response to the oxygenation conditions and nutrient feed composition is reported and the attendant effects of the nutrient additives on the morphology and physiology of the fungus are also described. MnP activity was found to increase by 58.16% in stationary batch cultures and an increased MnP productivity of 88.15% was observed after continuous operation with the SCMGR. The phospholipid rich production medium also stimulated intracellular organelles involved in respiration and protein synthesis, and increased bioﬁlm development radially across the membrane support matrix. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Microbial enzyme systems have been widely studied for both their ability to transform environmental pollutants during bioremediation and also for their potential commercial value in bio processing applications . Early research focused on both growth medium manipulation  and strain improvement  while whole cell bioreactors for large scale enzyme production have also been investigated with various modiﬁcations intended to either compensate for one or more of the rate limiting growth conditions  or to mimic the natural state of the organism [1,5,6]. This is of particular relevance to the aerobic, shear sensitive ligninase producing white rot fungi (WRF) such as the well-studied Phanerochaete chrysosporium. Industrial applications of ligninases range from the bioaugmentation of contaminated soil , bioremediation of industrial efﬂuents [7,8] for improving the digestibility of lignocellulosic feedstock for the production of platform chemicals and biofuels . The ligninolytic enzyme system of P. chrysosporium comprises a group of heme-containing peroxidases, MnP (EC 184.108.40.206) and lignin peroxidase (EC 220.127.116.11) and a copper containing phenoloxidase, laccase (EC 18.104.22.168). These ligninase enzymes [2,10,11], evolved for the degradation of the major structural polymer lignin,
∗ Corresponding author at: Council for Scientiﬁc and Industrial Research, CSIR Built Environment (RIS), Building 2a, PO Box 395, Meiring Naude Road, 0001 Pretoria, South Africa. Tel.: +27 12 841 2497; fax: +27 12 841 4054. E-mail address: [email protected]
(S. Govender). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.03.007
are produced by the fungi in response to severe limitation of C, N and S. The stereo-irregularity of lignin makes it very resistant to intracellular enzyme attack, so WRF instead degrades lignin extracellularly [2,12]. The conditions are referred to as ligninolytic and puriﬁcation and characterisation of MnP and lignin peroxidase (LiP) have been extensively reported [2,3,12]. The environmentally relevant fungi also produce oxidases and laccases that utilise the breakdown products of cellulose and lignin degradation as substrates for the production of extracellular H2 O2 from molecular O2 [7,8]. However, the ligninolytic enzymes which are produced during idiophasic metabolism are still dependent on the presence of certain growth and environmental conditions, where the onset is triggered by severe nutrient depletion. Furthermore, subsequent industrial application on a large scale has been hampered by the lack of suitable bioreactor conﬁgurations and an understanding of the physiological conditions of the fungus in response to nutrient limitation [6,10]. Conventional research strategy has involved innovative reactor design and geometry while usually using conventional feed and or modifying the nutrient composition in stationary batch cultures. Consequently, initial attempts at scale-up of continuous ligninase production, were limited to geometric scale-up of stationary cultures, and bioreactors adopting a strategy of immobilising the fungal biomass on a variety of porous supports, provided differing LiP and MnP yields. Innovative bioﬁlm systems [1,4,6,10] have also been developed based on the nutritional and physiological requirements of white rot fungi. However a membrane gradostat bioreactor (MGR), which was developed and characterised based on the observations of the white rot fungi in their native state, i.e., growing on a solid–air interface by
S. Govender et al. / Enzyme and Microbial Technology 46 (2010) 603–609 Table 1 Nutrient additives assessed for the development of a MnP production medium. Nutrient Asolectin Lecithin Tween 80 MnSO4
Concentration range 0.1–0.3% (w/v) 0.1–0.3% (w/v) 0.01–0.2% (v/v) 5–60 mg l−1
the metabolism of lignocellulose material, involves the immobilisation of the fungus onto capillary ultraﬁltration membranes [1,5,13]. Nutrient gradients resulting from radial, outward (tube to shell side) bioﬁlm development were exploited to provide both nutrient rich and poor zones. While this allowed for primary metabolism in the nutrient sufﬁcient zone, secondary metabolism is induced by starvation in the nutrient poor zones . This bioreactor system is ideally suited to mimic the fungus in nature but a low shear environment and adequate oxygenation for continuous operation remains crucial. In this study, a phospholipid rich feed supplemented with soybean phospholipids, Mn2+ and Tween 80 was applied to a MGR. Additionally the mode of oxygenation to the bioﬁlm (often a rate limiting step in aerobic bioreactors) was studied on batch bioreactors to optimise the O2 delivery which inﬂuenced the design of a continuous MGR. Using a SCMGR, the nutrient rich ‘production medium’ was compared with the widely used standard, ‘conventional’ feed of Tien and Kirk . The subsequent effects on MnP production, bioﬁlm morphology and ultrastructural physiology are reported using enzyme assays and electron micrographs. 2. Experimental 2.1. Microorganism, media and inoculum preparation P. chrysosporium ME446, was maintained on 2% malt extract agar plates, and incubated at 39 ◦ C for 10 days, prior to spore harvesting. The resulting conidial spores were prepared by suspending the spores in sterile deionised water followed by ﬁltration through sterile glass wool to free it of mycelia. Spore concentration was determined by measuring the absorbance at 650 nm . A commonly used or ‘standard’ nitrogen and carbon-limited medium , regulated at pH 4.5 by buffering with 20 mM Na-tartrate (Sigma, USA) solution was used as a control. However, the trace element solution used was deﬁcient in MnSO4 . Unless otherwise stated, all reagents were purchased from Sigma, USA. 2.2. Additive rich production medium Four nutrient additives (Table 1) were identiﬁed based on nutrient optimisation studies in other reports using ﬁlamentous fungi, and a concentration range was selected empirically. The anionic surfactant polyoxyethylene sorbitan monooleate, also known as Tween 80 (Fluka, South Africa), soybean asolectin and lecithin were sterilised by autoclaving and added to the standard growth medium described above. Filtered Mn2+ ions were added separately to the medium as MnSO4 (Merck, SA). The optimum concentration of each nutrient was deﬁned as the least amount of nutrient that caused the greatest relative increase in enzyme activity. 2.3. Stationary ﬂask cultures Laboratory scale experiments were initially performed in triplicate at 37 ◦ C in static 300 ml Büchner ﬂasks, where the fungus was introduced as a spore suspension (∼3.5 × 106 spores ml−1 ) into 30 ml of growth media. The extracellular ﬂuid was analysed daily for MnP and LiP activity, although only MnP activity is reported. Three modes of oxygenation were initially investigated with the design of a continuous MGR in mind, viz., oxygenation via the elevated side arm of the Büchner ﬂask, bubbling of O2 into the media and direct oxygenation using a sterile glass pipette suspended ∼4 cm above the mycelium. These stationary ﬂask cultures (SFCs) were ﬂushed with 100% O2 at 150 kPa for 2 min at the time of inoculation and every 72 h thereafter. 2.4. Membrane preparation Externally unskinned, anisotrophic polysulfone (PSU) capillary membranes were manufactured using the wet phase inversion process with the following components: PSU (polymer), NMP (solvent), methyl cellosolve (non-solvent additive), polyethylene glycol 600 (low molecular weight additive) and polyvinyl pyrrolidone
Fig. 1. Schematic illustration of the SCMGR: UF, ultraﬁltration capillary membrane; ST, silicone tubing; PP, permeate port; O2 , oxygen/air inlet. The complete process ﬂow diagram for membrane gradostat operation in continuous mode: P1 , peristaltic pump; AF, air ﬁlter; Po, outlet pressure guage; Ps, shell pressure guage; FF, feed ﬂask; PF, permeate ﬂask; V01 , valve (tube side); VOA , valve (shell side); V02 , valve (tube side); MBR, membrane bioreactor; O2 , 100% oxygen; P2 , peristaltic pump.
(40 kDa, high molecular weight additive) . The unique substructure morphology was designed to have low resistance to liquid transport and a large external surface area with well-deﬁned internal skin layers consisting entirely of closely packed and narrow-bore microvoids that extended from just below the internal skin layer of the membrane to the periphery. The regularly shaped microvoids opened into the membrane periphery due to a lack of an outer skin layer to bridge the cavities. 2.5. Bioreactor operation Before spore immobilisation the SCMGR was sterilised with 4% (v/v) formaldehyde and washed with sterile deionised H2 O . The SCMGR consisted of a single capillary UF membrane (OD 1.8 mm and length 200 mm) with a total membrane surface area of 1131 mm2 , encased in a borosilicate glass shell with an extra-capillary space (ECS) of 0.024 l. A schematic representation of the SCMGR and the process ﬂow diagram for continuous operation is illustrated in Fig. 1, with the attendant operating conditions described in Table 2. The SCMGR was dependent on aeration to generate a bioﬁlm and to stimulate ligninase production. The air-ﬂow regime (Fig. 1) consisted of continuously pumping in sterile air via a peristaltic pump and supplementing with intermittent ﬂushing of the reactor with 100% O2
Table 2 Dimensions and operating conditions of the SCMGR. Parameter
Membrane surface area Extra-capillary reactor volume Growth medium supply regime Permeate ﬂow rate Oxygen supply rate Air-ﬂow regime Temperature control
1131 mm2 ∼25 ml Constant ﬂux (4774.54 l m−2 h−1 ) 0.0100–0.0148 l d−1 1 vol. (vol ECS)−1 min−1 Transverse to capillaries Lagged shell (37 ◦ C)
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for 10 min every 12 h. The operational conditions selected for continuous operation were based on previous work with this gradostat system  and they were maintained constant for the duration of operation. Experiments were performed in duplicate. 2.6. MnP activity Mn(II) dependent peroxidase activity was determined by spectroscopically monitoring the oxidation of guaiacol . One unit of activity was deﬁned as the initial increase in absorbance at 465 nm of 1.0 per minute, and activities are reported in U l−1 . Productivity was deﬁned as the amount of product formed per unit reactor volume and was calculated as follows: P = A × D. Where P is the productivity [units × (active reactor volume)−1 d−1 ] = U l−1 d−1 . A is the activity of the enzyme in the reactor permeate = [units × l−1 ; where 1 unit is described as the catalytic ability to transform 1 mol of substrate in 1 min] = (U l−1 ). D is the dilution rate = [permeation rate (volume−1 )] = [(l d−1 ) × l−1 ]. 2.7. Intracellular enzyme activity Membrane sections with immobilised fungi were frozen in liquid nitrogen and stored at −70 ◦ C. Samples were ground in a mortar and pestle in the presence of liquid nitrogen. A sterile solution of sorbitol (1 M), HEPES (10 mM), EDTA (1 mM), pH 7.4 was added to obtain homogenous suspensions. They were then centrifuged at 5000 × g for 10 min. The supernatant was then collected and protein concentration measured, while mitochondria and endoplasmic reticulum (ER) development were estimated by marker enzyme assays. Succinate dehydrogenase (SSD), a marker for the mitochondria was determined spectrophotometrically by measuring the rate of reduction of the tetrazolium salt (MTT) resulting from the oxidation of succinate to fumarate. Cytochrome c oxidoreductase (COX) a marker for the ER was determined spectrophotometrically by measuring the reduction of cytochrome c [14,15]. 2.8. Scanning electron microscopy For scanning electron microscopy (SEM), PSU membrane sections were ﬁxed for 2 h in 5% (v/v) glutaraldehyde with 0.1 M phosphate buffer at 4 ◦ C and pH 7.5, after which the sections were dried in water:ethanol mixtures of 20, 40, 60, 80 and 100% ethanol. Cross-sections of the membranes were prepared by freeze fracturing using liquid N2 . Samples were examined using a Hitachi S-570 SEM (Japan). 2.9. Transmission electron microscopy Fungal tissue to be analysed by transmission electron microscopy (TEM) was sliced, collected and placed in fresh glutaraldehyde ﬁxative. After ﬁxation in glutaraldehyde the tissue was washed for at least 60 min in two changes of 0.05 M cacodylate buffer. Post-ﬁxation was then carried out using osmium tetroxide. After the required length of time in osmium tetroxide the material was then rinsed several times in 0.05 M Na-cacodylate buffer in a fume cupboard. At least two washes of 30 min each are required to ensure that all the osmuim tetroxide was removed before dehydration in a graded alcohol series. The material was then embedded in Spurrs resin. Sectioning was done using a LKB ultramicrotome, post-ﬁxed with uranyl acetate and Pb citrate and viewed with a JEOL 1210 TEM.
3. Results and discussion 3.1. Development of an additive rich production media Initial screening of the effect of nutrient additives (Mn2+ , Tween 80 and soybean derived phospholipids) and oxygenation was performed on a batch scale using stationary ﬂask cultures, where there were fewer variables involved and which were much easier to operate, control and analyse than a continuous bioreactor. A Büchner ﬂask was used for batch studies where a minimum ratio of culture to reactor volume of 1:10 was maintained. Oxygen was already shown to be a prerequisite for high ligninolytic activity [2,3] so one of the most important environmental design considerations inﬂuencing ligninase production in bioreactors is the provision of adequate O2 to the fungus. The effects of the mode of oxygenation (via a side arm, bubbling into media and direct ﬂushing ∼4 cm above the mycelium) on MnP activity is shown in Fig. 2. Since the consensus regarding the mode of oxygenation to P. chrysosporium in both SFCs and bioreactors in the literature is not clear this study has shown that gentle oxygenation tangential to the bioﬁlm gave the highest MnP activity (112 U l−1 ). Simply ﬂushing the ﬂask to avoid disturbing the
Fig. 2. The impact of O2 delivery to SFCs using the conventional ligninase producing growth media. N = 3.
stationary culture medium is comparatively ineffective (39 U l−1 ), most likely due to the low solubility of the gas while gentle bubbling of 100% O2 for 2 min every 72 h was not optimal either (66 U l−1 ). Tangential aeration just above the mycelium was also able to overcome the primary resistance at the gas–liquid interface and was a major factor inﬂuencing the design of the MGR. Screening of low cost nutrient additives with potential to stimulate or increase enzyme production is outlined in Table 1. MnSO4 has been shown in batch cultures  to induce MnP production while repressing synthesis of the LiP isoform. A concentration dependence on MnSO4 was observed in Fig. 3A. Mn(II) is known to selectively induce the production of the MnP isoform and can act as a scavenger of toxic superoxide radical ions (O•2− ), replacing the micromolar levels of superoxide dismutase which functions in other O2− tolerant organisms . Peak MnP activity was obtained with 60 mg l−1 MnSO4 . Tween 80 has been reported to stabilise enzyme activity against shear forces during agitation [2,17] and to both emulsify oleic acid supplements and stabilise ligninase activity in submerged agitated cultures . Although nutrient feed supplementation with Tween 80 surfactants was not detailed in reports based on static cultures, it was nevertheless reasoned that since Tween 80 is also a known adjuvant  it could also modify the effects of other nutrient additives or supplements in the culture medium. The changes in MnP activity (Fig. 3B) with Tween (optimum at 0.1%) suggests that it has multiple functions in the culture media and is more than just a stabiliser in stirred cultures but can also act as an adjuvant and or an emulsiﬁer. A few reports have shown a link between soybean phospholipids in the growth media and the production of aryl metabolites with white rot fungi such as Bjerkandera adjusta . The increase in MnP activity with soybean derived asolectin and lecithin is shown in Fig. 3C and D. This also correlates with the ﬁndings of other studies where a relationship between phospholipid stimulation and ligninase activity was shown [11,15]. The optimum concentration ranges of soybean phospholipids, Tween 80 and 60 mg l−1 Table 3 Intracellular and extracellular enzyme activities associated with nutrient feed manipulation and continuous SCMGR operation. Nutrient cofactor
MnP (U l−1 )a
SDH (U g−1 )b
COX (U g−1 )b
Control 0.3% (w/v) asolectin 0.1% (v/v) lecithin Tween 80 Production medium Run A Run B
98 134 155 101 185 16.5 31.1
5.0 7.2 9.1 5.3 9.9 2.7 3.3
22 31 37 25 43 12.1 30.5
Peak MnP activities extrapolated from Figs. 4 and 5. Data expressed as units of enzyme activity per gram of intracellular protein.
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Fig. 3. The inﬂuence of supplementing the conventionally used growth media with nutrient additives and the effects on MnP production. (A) Mn2+ ions, (B) Tween 80, (C) asolectin, and (D) lecithin. Each data point represents the mean for triplicate cultures while the standard error was always less than 10% of the mean. Error bars not shown. N = 3.
MnSO4 were compared (Fig. 4) and calculated (Table 3) based on their abilities to enhance ligninase activity with a bias towards MnP production which was found to be far more reproducible in terms of both enzyme production and stability than LiP. From the data in Fig. 4a production medium was developed based on the nutrient additives Tween 80 [0.1%, w/v], asolectin [0.35%, w/v] and lecithin [0.2%, w/v]. MnP activity increased by 58.16% using the nutrient modiﬁed production medium, with a peak observed after 6 days of growth in stationary batch cultures. The importance of Tween 80 could also be related to its ability to alter membrane permeability , thus increasing the bioavailability of the other nutrient cofactors (asolectin and lecithin) which stimulate the intracellular organelles involved in protein biosynthesis.
Fig. 4. MnP activity proﬁles due to combination testing of optimised nutrient additives for the development of a production medium. The error associated with each point without a visible error bar is less than 6% of the value of that point and said error bar is smaller than the attendant symbol. N = 3.
3.2. Continuous MnP production in a membrane gradostat reactor The optimised nutrient supplemented medium, tested using SFCs was developed for use as a production medium in the membrane gradostat bioreactor and to assess the feasibility of using a separate growth and production medium for enhanced MnP production. Tests were initially carried out on disposable SCMGRs which were developed for rapid sequential destructive sampling of the polymeric membrane associated bioﬁlm. The ﬁrst investigation (run A) involved continuous operation using only the conventional nutrient system . In the second experiment (run B), the phospholipid rich production medium was used instead. The conditions employed for run A were used initially for the third experiment (run C), and this feed was replaced with the phospholipid rich feed as used in run B, after 3 days of continuous operation or after a bioﬁlm layer was observed and just before the onset of ligninase activity. The three runs (A–C) each lasted an average of 23 days before they were stopped due to the detection of fungal growth (P. chrysosporium) in the feed ﬂask and permeate tubing. However, no contamination was observed on the immobilised bioﬁlm surface or within the asymmetric pores. This was attributed to the exclusion properties of the internally skinned UF membrane and the sterilisation step using 4% (v/v) CH2 O which sterilised the bioreactor in both the aqueous and gaseous phase. Traces of MnP activity were detected in the reactor permeate after 4 days of continuous operation (Fig. 4). LiP activity was not detected, but this most likely due to the relatively small reactor volume available for bioﬁlm development and the use of the weakly ligninolytic strain ME446 [2,17], rather than exclusively a MGR shortcoming in either design strategy, mode of operation or a combination thereof. Uniform growth of the membrane immobilised bioﬁlm during run B using the nutrient supplemented medium resulted in a peak productivity of 31.12 U l−1 d−1 . This was an
S. Govender et al. / Enzyme and Microbial Technology 46 (2010) 603–609
Fig. 5. MnP productivity proﬁle during continuous SCMGR operation. N = 2.
88.15% increase compared to the peak of 16.54 U l−1 d−1 after 7 days of operation in run A. Moreover replacing the conventional feed after 8 days of continuous operation in run C with the production medium developed in this work, did not result in a signiﬁcant increase in enzyme productivity (peak of 20 U l−1 d−1 after 18 days) as compared to the 31.12 U l−1 d−1 after 3 days. Assuming the arbitrarily chosen operating conditions were successfully maintained constant during all three investigations, the higher MnP productivity with run B was also thought to be inﬂuenced by the initial germination and bioﬁlm development process which occurred in the presence of the nutrient additives in the feed. Supplementing the feed with phospholipids and Tween 80 later in the cycle was not as effective as germination and exploratory growth in the presence of these nutrient cofactors. For optimal enzyme production the modiﬁed phospholipid rich feed should ideally be incorporated into the initial spore germination stage and not just when a suitable bioﬁlm thickness was observed or when the stationary phase was estimated. This was contrary to the practice of using separate germination and production media . The pump ﬂow rate was maintained constant so as to minimise the inﬂuence of hydrodynamic parameters on the shear sensitive MnP enzyme production pathway and bioﬁlm development.
Fig. 6. Permeate ﬂow rate during SCMGR operation for runs A (conventional feed), B (production medium) and C (alternating between the feed systems used in runs A and B). N = 2.
The C and N ratio of the feed systems in runs A and B were also kept constant for the duration of the run enabling bioﬁlm proliferation in the nutrient rich regions of the bioﬁlm with the older biomass being pushed outwards towards the O2 richer zone (Fig. 7). The SCMGR was run at constant ﬂux so as to minimise the effects of hydrodynamic parameters on enzyme production and bioﬁlm
Fig. 7. Bioﬁlm development along the capillary membrane after destructive sampling for SEM. (A) Surface colonisation and development after sloughing off of older mycelium (bar = 29 m), (B) mature differentiating hyphae (bar = 138 m), and (C) bioﬁlm growth (25 days) on the capillary membrane. The brown mycelium is associated with ligninolytic production/activity, while the bare regions are indicative of sloughing off of older mycelium, particularly near the O2 inlet port.
S. Govender et al. / Enzyme and Microbial Technology 46 (2010) 603–609
Fig. 8. TEM micrographs showing the effect of the optimised production medium on mitochondria (m) proliferation. (A) Cells from fungi grown during run A (magniﬁcation = 10 000×) and (B) cells grown during run B (magniﬁcation = 12 500×).
development. This also led to a gradual decrease in the permeation rates (Fig. 6) which although led to a longer residence time of permeated nutrients within the bioﬁlm and hence a more concentrated permeate, the total volume of permeate collected at the end of the day for reproducible analysis was very low. 3.3. Bioreactor physiology and productivity Visual assessment of the capillaries showed much thicker and darker bioﬁlm on the membrane used in run B as compared to runs A and C. The dark brown colour of mycelium (Fig. 7) has also been associated with the production of ligninase enzymes in the presence of excess trace elements in ﬂask cultures [1,2]. Different growth phases, i.e., exponential, stationary and decline phases were observed radially across the bioﬁlm (Fig. 7A and B) after sectioning along the length of the capillary membrane. This observation was consistent for all three runs and is a typical feature of membrane gradostats. Once older mycelium, especially portions growing close to the O2 inlet port was sloughed off the membrane, new growth occurred and exploratory hyphae were observed in Fig. 7A. The denser brown bioﬁlm were conﬁrmed to be mature mycelial mats in idiophasic growth (Fig. 7B). Ultra structural analysis of the mycelium suggested that runs B and C encouraged bioﬁlm development. This was supported by the observed trends in the permeation rate decrease in Fig. 6, where runs B and C showed a much more substantial decrease in the permeation rates which is most likely attributed to the heavier biomass development (related to growth with the nutrient additives) which limited the permeate ﬂux through the membrane. Furthermore, an increase in MnP activity also coincided with corresponding increases in SDH and COX activity, which are intracellular markers for the mitochondria and ER respectively (Table 3). The intracellular COX and SDH activities were reported in relation to the intracellular protein levels (Table 3). In bioﬁlm sections from run B (using the modiﬁed medium) COX and SDH activities were found to be 152.07% and 22.2% higher respectively than with bioﬁlm from run A (using conventional feed). This supported the observations of studies [15,19] where phospholipid additives enhanced intracellular marker enzyme activity for batch cultures using stationary ﬂasks were reported. TEM analysis of the bioﬁlm from run B, showed much higher numbers of mitochondria per cell using the ‘production medium’ than bioﬁlm treated with the conventional feed (Fig. 8). The ultrastructural evidence also correlates with the increased SDH activity (Table 3) which has been shown to stimulate energetic metabolism in vivo. A direct correlation between ER proliferation using TEM and COX activity was inconclusive due to poor resolution of the ER with the relatively insensitive section staining technique employed. The intracellular enzyme activities however do suggest a correlation
between proliferation of intracellular organelles associated with MnP production, transport and respiration (mitochondria) and protein synthesis (ER). It has also been suggested that in the presence of phospholipids enzymes would be secreted not as free enzymes but as enzyme–phospholipid complexes . The advantage for continuous ligninase production is that the destructive action of these enzymes on the cytoplasm would be decreased. Addition of soybean phospholipids has also been shown to cause an enrichment of phospholipids and lipids and an increase in the ﬂuidity of the inner membrane in P. chrysosporium INA-12 grown in stationary ﬂask cultures [15,22]. Thus the increased production of MnP using the MGR and the modiﬁed production medium can be correlated with changes in membrane composition, bioﬁlm proliferation and protein biosynthesis. 4. Conclusions Stationary ﬂask cultures operated in batch mode were well suited for the investigation of O2 delivery and the addition of nutrient additives to the widely used conventional growth media  used for ligninase production with P. chrysosporium. Oxygenation tangential to the mycelium for 2 min every 72 h resulted in higher enzyme activity (MnPmax = 112 U l−1 ) than both elevated side arm oxygenation (MnPmax = 39 U l−1 ) and bubbling O2 into the growth media (MnPmax = 66 U l−1 ). Results of said investigation were a primary consideration during the design and construction of subsequent membrane gradostat bioreactors. Furthermore, all four nutrient additives (Mn2+ , Tween 80, asolectin and lecithin) were found to enhance MnP activity both individually and when combined to form an optimised ‘production media’. The optimum concentrations were calculated by correlating the largest increase in MnP activity with the corresponding lowest amount of nutrient additive used. A combination of the ‘optimum’ amounts into a new production medium (for continuous MGR operation) resulted in a 58.16% increase in MnP activity compared to the conventional medium. Said production medium was also effective when tested on a SCMGR. MnP productivity increased 88.15%, while no adverse effects with reactor operation or bioﬁlm growth were found. Using a SCMGR, no beneﬁt was found in alternating between a separate growth and production medium as discussed in other reports. The morphology of the immobilised bioﬁlm was monitored using SEM and hyphal proliferation and differentiation was attributed to the nutrient manipulation in the production medium. Intracellular marker enzymes and ultrastructural hyphal analysis were used to correlate the physiological response of the fungus to the additive rich feed and MnP productivity. COX and SDH, marker enzymes for the endoplasmic reticulum and the mitochondria showed increased activities by 152.07% and 22.2% respectively. A link between SDH activity and intracellular mitochondria pro-
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