Process Biochemistry 36 (2000) 149–155

Fermentation and downstream processing of lipase fromAspergillus terreus

Ruchi Gulati, R.K. Saxena *, Rani GuptaDepartment of Microbiology, Uni6ersity of Delhi (South Campus), Benito Juarez Road, New Delhi 110021, India

Received 6 December 1999; received in revised form 12 May 2000; accepted 27 May 2000

Abstract

Fermentation behaviour of Aspergillus terreus lipase was studied in a 10 l fermentor. Lipase production was enhanced to 14 200U l−1 in 54 h in the fermentor as against 7000 U l−1 in 96 h in shake flasks under optimised nutritional conditions. A 2.4-foldincrease in specific activity (16.2 U mg−1 protein) was also attained. Inoculum density, dissolved oxygen levels and agitation werethe major controlling factors. A two-step cost-effective downstream processing methodology comprising of an aqueous two-phasesystem (ATPS) of polyethylene glycol (PEG) and phosphate was devised. This procedure resulted in a 12-fold purification of thelipase with 100% yield in less than 1 h. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Lipase; Fermentation; Downstream processing; Aqueous two phase system

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1. Introduction

Lipases or triacylglcerol hydrolases (E.C. 3.1.1.3)catalyse the hydrolysis of fats to di- and monoglyce-rides and free fatty acids [1]. They have considerableand versatile industrial potential as catalysts of hydrol-ysis, synthesis, trans-esterifications and optical resolu-tion [1,2]. Although numerous papers have beenpublished on fermentation process for large scale pro-duction of lipase but these are mostly with bacteria[3–6] or yeasts [7,8]. The fermentation processes of onlya few fungal lipases e.g. Rhizopus delemar [9], Asper-gillus oryzae [10], Mucor meihei [10] and Geotrichumcandidum [11] has been characterised.

Lipase production by Aspergillus terreus in shakeflasks under various nutritional conditions was reportedearlier [12]. A. terreus produces a novel thermostablelipase [13] capable of carrying out deacetylation ofpolyphenolic compounds at the ‘ortho ’ position, aproperty unique to this lipase alone [14]. This enzyme isalso able to carry out synthesis of a variety of esters foruse in flavour development, cosmetics [12] and as bio-

surfactants [15]. Owing to its commercial importance, abatch fermentation process for A. terreus lipase produc-tion using a 10 l fermentor to obtain higher enzymeyields in less time is reported. In addition, a cost-effec-tive methodology was developed for downstream pro-cessing of the lipase using an aqueous two phasesystem.

2. Materials and methods

2.1. Microorganism and production medium

A. terreus (RKS101) was maintained on potato dex-trose agar slants at 4°C in a B.O.D. incubator. Lipaseproduction was carried out in the production mediumas described previously [12]. The medium consisted of(in g l−1 distilled water) NaNO3, 2; KCl, 0.52;MgSO4 · 7H2O, 0.52; KH2PO4, 1.52; Cu(NO3)2 ·3H2O, 0.001; FeSO4 · 7H2O, 0.001; ZnSO4 · 7H2O,0.001; glucose, 2.0; casein, 1.0; and corn oil, 2.0% v/v.Calcium (CaCl2 · 2H2O) and magnesium (MgCl2) ionswere added to a final concentration of 1.0 and 1.5 mM,respectively. Corn oil was emulsified using 10% w/vgum acacia.

* Corresponding author. Tel.: +91-11-6886559; fax: +91-11-6886427/5270.

E-mail address: micro@dusc.ernet.in (R.K. Saxena).

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R. Gulati et al. / Process Biochemistry 36 (2000) 149–155150

2.2. Fermentation conditions

Fermentation was carried out in a 10-l fermentor(New Brunswick Sci. Inc., USA and Bioflow IV model)using 5 l of the optimised production medium [12]. Themedium was steam sterilised in situ except for glucose,which was sterilised separately and added along withthe inoculum. The medium was inoculated with a sporesuspension (5×107 spores per 50 ml) for 5 l of theproduction medium of a 96-h-old culture of A. terreus.Standard operating conditions for the fermentor runwere, temperature, 37°C; agitation, 250 rpm; non-con-trolled pH (initial pH 9.0); and airflow rate, 1 vvm.Samples were drawn periodically during the run andanalysed for extracellular lipase activity, protein con-tent and biomass. The mycelial biomass was separatedfrom the culture broth containing extracellular lipase byvacuum filtration on Whatman filter paper no. 1. Devi-ations from any of these conditions are mentioned asappropriate. Lipase activity was measured spectropho-tometrically with a p-nitrophenyl palmitate assay [16]and by titrimetry using olive oil as the substrate [17].The protein content was analysed by the Lowry method[18]. The biomass was estimated by filtering the con-tents of the culture broth through a pre-dried andpre-weighed Whatman filter paper no. I and drying at80°C till a constant weight was achieved.

The following factors were studied in order to opti-mise lipase production under batch fermentation:1. Inoculum levels: the effects of inoculum levels rang-

ing from 5×105 to 5×107 spores per 50 ml of theproduction were examined. Fermentation was car-ried out under uncontrolled dissolved oxygen condi-tions and all other operating parameters wereindicated above.

2. Controlled dissolved oxygen conditions: lipase pro-duction was carried out under two different percent-ages of oxygen i.e. 20 and 40% after the dissolvedoxygen (DO) fell below a critical value of 10–15%during the run. A computerised agitation–aerationcascade was employed to maintain a DO concentra-tion above these levels. The air-flow-rate was variedbetween 1 and 2 vvm and agitation between 250 and400 rpm to achieve the saturation levels.

3. Agitation rates: the effect of varying initial agitationrates ranging from 250 to 400 rpm at an interval of50 rpm were studied on lipase production. The DOwas controlled above 20% saturation throughoutthe production period.

2.3. Downstream processing of lipase from fermentationbroths

Following fermentation, the culture broth was har-vested by separating the fungal mycelium from theculture filtrate using vacuum filtration. The culture

filtrate containing extracellular lipase was subjected toclarification by activated charcoal to remove pigmentsand other colloidal matter. The clarified filtrate wassubjected to purification by partitioning in an aqueoustwo phase system (ATPS). The ATPS comprised ofpolyethylene glycol (PEG) and phosphate. The phasesystem was prepared from stock solutions of PEG-6000(100% w/w) and phosphate (50% w/w). The phosphatestock solution comprised of a mixture of K2HPO4 andNaH2PO4 in appropriate ratios to produce the desiredpH. The selection of appropriate concentrations ofPEG and phosphate for phase development was doneaccording to the binodials of PEG-6000 and phosphateprepared according to Albertsson and Tjerneld [19].

For partition experiments, 2.5 ml of crude lipase wasadded to the phase system (10-ml-volume). After 20min of gentle shaking at room temperature, the mixturewas centrifuged to attain phase separation and parti-tioning of enzyme between the phases. Gentle shakingtime (20 min) was selected after standardisation of thetime to obtain maximum partition coefficient for lipaseand thus to obtain the steady state conditions. Sampleswere withdrawn from top and bottom phases andanalysed for lipase activity by p-nitrophenyl palmitateassay [16] and for total protein by the Biuret method[20]. The partition coefficient log K was calculated ac-cording to the following equation:

K=Total lipase per protein in the top phase

Total lipase per protein in the bottom phase

The following factors were studied in order toachieve maximum log K value for lipase and purifica-tion folds (minimum log K for total protein):1. different combinations of PEG and phosphate;2. varying concentrations of PEG-6000;3. effect of ionic strength of the phase system (as a

function of added NaCl [% w/w]); and4. effect of pH.

A two step procedure was devised to ensure recyclingof the phase components for a cost-effective process.

3. Results

It was reported earlier [12] that under shake flaskconditions, A. terreus produces 7000 U l−1 of lipase at37°C, pH 9.0 after 96 h at 250 rpm. With a view toenhance lipase yields further, the process was optimisedin a 10-l fermentor by manipulation of various fermen-tation parameters.

Fig. 1 presents lipase production at various inoculumlevels. It was observed that higher inoculum levels of1×107/5×107 spores per 50 ml for 5 l of the mediumsupported a maximum lipase production of 9000 U l−1

in 72 h. In contrast to this, lower inoculum levelssupported lower lipase production i.e. 7000 U l−1. In

R. Gulati et al. / Process Biochemistry 36 (2000) 149–155 151

addition, the time period was extended beyond 78 h.Growth dependent lipase production was observedwhich was linked to the sugar utilisation profile of theorganism (Fig. 2). At higher inoculum levels, glucose(initial level 2 g l−1) was consumed much faster by A.terreus and thus a shift towards utilisation of corn oil

Fig. 3. Effect of DO levels on A. terreus lipase fermentation.

Fig. 1. (a) Influence of inoculum size on lipase fermentation by A.terreus ; (b) specific activities of lipase of A. terreus at various inocu-lum densities.

and hence lipase production was achieved at an earlystage. However, at lower inoculum densities, due toslower growth and hence delayed utilisation of sugar,lipase production was delayed. Further high specificactivities were observed with high inoculum densities(Fig. 1). An inoculum level of 1×107 spores per 50 mlwas therefore selected for further studies. A maximumspecific growth rate (mmax) of 0.05 h−1 was obtained.Foaming was not a problem due to the presence of cornoil in the medium. The pH of the medium fell to only1.290.2 during the fermentation run. DO was notcontrolled during the run and was allowed to fallgradually as a result of growth.

The effect of varying DO levels is shown in Fig. 3.Lipase production of 12 000 U l−1 was achieved in 60h when the DO was maintained above 20% saturationfor the remainder of the fermentation run after it fellbelow a critical concentration of 10–15%. It was alsoobserved that increasing the DO level (40% saturation)did not further enhance lipase production thereby indi-cating that maintaining DO level above a certain criti-cal/limiting value was important for lipase productionrather than the saturation percentage of DO.

Both biomass and lipase production were affected byagitation rates (Fig. 4a and b). Lipase production fur-ther increased to 14 200 U l−1 and a reduction infermentation time to 54 h was observed at agitationspeed of 300 rpm (Fig. 4). Thus an increase in lipaseproduction on increasing agitation could be due toincreased oxygen transfer rate, increased surface area ofcontact with the media components or better dis-persability of the oil substrate. A specific activity of16.2 U mg−1 protein was achieved at agitation rate of300 rpm. There was no enhancement in lipase produc-tion on increasing the agitation rates further. Howeverat agitation rates of 400 rpm, there was a reduction ingrowth as well as lipase production due to shearingstress on the organism as evidenced by fragmentationof the mycelium, which was observed microscopically.

Fig. 2. Biomass and sugar utilisation profiles during lipase fermenta-tion with low and high inoculum densities. For 5×107 spores per 50ml: residual sugar (closed circle), biomass (closed square); 5×105

spores per 50 ml: residual sugar (open circle), biomass (open square).

R. Gulati et al. / Process Biochemistry 36 (2000) 149–155152

Fig. 4. (a) Effect of agitation rates on A. terreus lipase fermentation;(b) biomass profiles of A. terreus at different agitation rates duringlipase fermentation.

Fig. 5. Effect of PEG-6000 concentrations on partitioning and purifi-cation of A. terreus lipase in aqueous two phase system.

lipase was partitioned into the top PEG phase with100% lipase yields and purification 2.07-fold (Table 1).A strong activation of lipase in the presence of PEG wasalso observed. PEG-6000 concentrations were variedfrom 40 to 70% w/w and at concentrations above 50%w/w, the lipase essentially remained in the top phase dueto the high affinity for PEG. Purification was doubled atPEG concentration of 50% w/w thereby indicating thatmost of the undesired proteins partitioned into thebottom phosphate phase (Fig. 5). This concentrationwas thus selected for further studies.

On addition of NaCl (% w/w) as a function of ionicstrength to the phase system, it was observed thatamong the various concentrations of NaCl (0.5–10%w/w) tested, at concentrations of 0.5% w/w, purificationwas increased significantly from 4.37 to 7.03-folds (Fig.6). However, further increase in concentration of NaCldecreased log K for lipase.

The effect of pH on partitioning showed that at thelower pH of 4–6, lipase was partly partitioned into bothphases. At pH 7.0, A. terreus lipase specifically parti-tioned into the top phase (Fig. 7). At high pH, all thelipase partitioned into the top phase, but specific activitydecreased due to the fact that some of the contaminat-ing proteins get partitioned to the top phase. Thus fromthe above studies, a phase system comprising of PEG,50% w/w; phosphate, 25% w/w and NaCl, 0.5% w/w atpH 7.0 was selected.

3.1. Downstream processing of lipase from A. terreus

Following fermentation, clarification of the lipasebroth was achieved by treating with 1% w/v activatedcharcoal, which removed pigments and colloidal matter.Residual lipase activity in the clarified broth was as-sayed by the p-nitrophenyl palmitate assay and it wasfound that charcoal did not adsorb any lipase as 100%residual activities were observed in the clarified broth.The clarified lipase broth was subjected to purificationby partitioning in an aqueous two phase system. Vari-ous factors were studied in order to maximise thepartition coefficient log K and specific activity to achievegreater purification folds and yields of the enzyme.

Among various combinations of PEG and phosphatetried, in a PEG–phosphate system of 60.25% w/w, total

Table 1Effect of different combinations of PEG-6000 (w/v) and phosphate on partitioning of A. terreus lipase

Total lipase (U)ATPS log K (lipase) Specific activity (U mg−1 protein) Purification (fold)

Top phasePhosphatePEG Bottom phaseBottom phase Top phase

–60 2.0725 7.0939.40 – 1.603.6540 2.10 1.0714.58 12.76 0.0540

30 2.5050 1.5810.73 18.39 0.20 5.393.4222.94Crude lipase

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Fig. 6. Effect of varying ionic strengths (% w/w NaCl) on lipasepartitioning and purification.

added. Lipase at this pH value was partitioned into thebottom phase. A significant increase in purification to11.0-fold with 100% lipase yields was achieved (Table2). The process was scaled up to 1 l of the fermentationbroth. A schematic representation of the whole fermen-tation process and downstream processing of A. terreuslipase is shown in Fig. 8. This could be completedwithin 60 h.

4. Discussion

The development of a batch fermentation process forproduction of lipase from A. terreus is described, em-ploying manipulation of various factors including in-oculum density, oxygen tension and agitation rates.When compared with earlier studies in shake flasks [12],lipase production was enhanced to 142 000 U l−1 inthe fermentor and a 2.4-fold increase in specific activityof lipase. Most importantly, time period for the produc-tion process was significantly reduced from 96 h (inshake flasks) to 54 h in the fermentor.

In common with various other organisms like R.delemar [9] and Acinetobacter radioresistens [5] andCandida rugosa [21], lipase production by A. terreus wasa growth-associated phenomenon. In contrast to this,lipase production by Pseudomonas aeruginosa [6] wasnot growth associated as lipase was induced only in thelate exponential phase and reached a peak in the latestationary phase. Lipase production was optimal athigh inoculum densities of A. terreus, which was di-rectly related to sugar utilisation. However, in the caseof R. delemar lipase [9], lipase production was reducedat high inoculum densities.

Further enhancement in lipase production rate aswell as yield was observed on controlling dissolvedoxygen levels above a saturation level even thoughgrowth remained the same. Here too, the DO level wasnot important if it was maintained above a certaincritical point (i.e. above 20% saturation) throughout thefermentation run as similar production levels were ob-

Fig. 7. Lipase partitioning in aqueous two phase system as a functionof pH.

To make the whole process more cost-effective, recy-cling of the phase components was accomplished bydevising a two step ATPS. The first step involved lipaseextraction in the top PEG phase using ATPS selectedabove. The second step comprised of back extraction ofthe lipase into a fresh bottom phase, to allow for PEGrecycling. For this, the primary phosphate phase wasremoved and to the PEG phase (diluted to 30% w/w)containing lipase, fresh phosphate phase (pH 5.0) was

Table 2Partitioning and purification of A. terreus lipase using a ‘two step aqueous two phase system’

Step Total lipase (U) Specific activity (U mg−1 protein) Purification (fold)

–2250Crude lipase 3.42

First step ATPS (pH 7.0)Topa 25.013900 7.31

– – –Bottom

Second step ATPSb (pH 5.0)–Top ––

3900 33.65Bottom 11.01

a Primary PEG phase.b Primary PEG phase was diluted to 30% w/w for the second step and fresh phosphate (pH 5.0) was added.

R. Gulati et al. / Process Biochemistry 36 (2000) 149–155154

Fig. 8. Schematic representation of production and downstream processing of A. terreus lipase.

tained at 40% saturation level of DO. Thus the criticaldemand for oxygen was higher for lipase productionthan for growth. Similar results have been obtained inthe case of P. aeruginosa [6] where maintaining DOlevels above a critical level enhanced lipase productionin comparison to a situation where DO levels wereuncontrolled.

Both growth and lipase production were affected byagitation rates even though DO was controlled atthe same level. Further increase in lipase productionas well as reduction of fermentation time on increas-ing agitation to 300 rpm was due to an increase inoxygen transfer rates. Thus the modelling of aerobicfermentations in terms of oxygen transfer rates is moreadequate rather than in terms of DO levels as sup-ported by Chen et al. in their studies on A. radiore-sistens [5].

The use of aqueous two phase system for purificationof proteins and other bio-molecules is gaining signifi-cance due to the smaller time involved and the higheryields and purification levels achieved [22]. The twostep procedure devised for A. terreus lipase has theadvantage of obtaining 11-fold purification with 100%lipase yields in less than 1 h. Further it allows forrecycling of the phase components thus making thewhole downstream process more cost-effective. Thusthe downstream processing methodology is fast andsimple and involves a smaller number of steps (onlytwo steps). Further it gave higher lipase yields in com-parison to conventional purification procedures and isthus very cost-effective. Similar results have been ob-tained in the case of M. miehei lipase using a PEG saltATPS with a yield of 80 and 69% purification of thelipase [23].

R. Gulati et al. / Process Biochemistry 36 (2000) 149–155 155

5. Conclusions

The whole process of production and downstreamprocessing of lipase by A. terreus is cost-effective aswell as time-saving. The overall fermentation anddownstream process takes less than 60 h. Higher lipaseyields were obtained both in terms of production andpurification methodology adopted. This is importantfrom a commercial viewpoint as has already beenshown by many industrially important applications ofA. terreus lipase.

Acknowledgements

The authors wish to thank the Department of Bio-technology, Govt. of India for financial assistance.Ruchi Gulati wishes to thank University Grants Com-mission for grant of senior research fellowship. Thehelp provided by Satish Kumar in running and main-taining the fermentor is duly acknowledged.

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