Increased Hepatitis B surface antigen production by recombinant Aspergillus niger through the...

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Increased Hepatitis B surface antigen productionby recombinant Aspergillus niger through the optimizationof agitation and dissolved oxygen concentration

Emmanuel R. James & Willem H. van Zyl &Johann F. Görgens

Received: 8 October 2006 /Revised: 15 December 2006 /Accepted: 15 December 2006 / Published online: 17 February 2007# Springer-Verlag 2007

Abstract The capacity of the filamentous fungi Aspergillusniger to produce and assemble complex immunogenic viralproteins into virus-like particles (VLPs) in batch culturewas enhanced by optimizing the bioprocessing parameters,agitation intensity and dissolved oxygen (dO2) concentra-tion. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD)were employed to evaluate the interactive response patternbetween parameters and their optimum combination. Therecombinant hepatitis B surface antigen (HBsAg) was usedas a model VLP system to determine the effect of theseparameters on biomass yield, fungal morphology, HBsAgproduction and bioreactor kinetics. The response surfacemodel predicted optimum cultivation conditions at anagitation of rate of 100 rpm and a dO2 concentration of25%, obtaining highest intracellular membrane-associatedHBsAg levels of 3:4 mg l�1

culture. HBsAg production levelswere increased tenfold compared to yields obtained inshake flask cultivation. Although hepatitis B VLPs mostlyaccumulated intracellularly, optimal bioreactor conditionsresulted in significant HBsAg release in culture supernatant.These results compare favourably with other recombinantVLP systems in batch culture, and therefore, indicate asubstantial potential for further engineering of the A. nigerproduction system for the high level of intracellular andextracellular VLP production.

Keywords VLP production . HBsAg . A. niger .

Dissolved oxygen concentration . Agitation intensity

Introduction

Vaccines have been one of the most far-reaching andimportant public health initiatives in recent times. Consid-erable attention is being paid to the development ofrecombinant microbial expression systems as hosts forimmunogenic virus-like particle (VLP) production, for usein safe and effective vaccines (Hansson et al. 2000). Thefilamentous fungi Aspergillus niger is one host that hasgained much interest for expressing mammalian and highereukaryotic gene products, due to several potentiallyadvantageous characteristics. Aspergillus species haveexpression–excretion systems with an exceptional highprotein secretion capacity (Jeenes et al. 1991; Van denHondel and Punt 1991). Recent advances in fungal geneticshave enabled expression of several high value proteins instable constructs (Upshall et al. 1987; Gouka et al. 1997;Ward et al. 1995; Gwynne et al. 1987; Davies 1994).Filamentous fungi have the ability to express heterologouseukaryotic proteins in a correctly folded and functionalform. They are also robust during cultivation and can growon various substrates and organic compounds (Jeenes et al.1991; Van den Hondel and Punt 1991), resulting incultivation methods that are substantially cheaper thanvarious other expression systems. These advantageouscharacteristics of filamentous fungi were confirmed whenPlüddemann and van Zyl (2003) demonstrated the ability ofthe filamentous fungi A. niger to produce VLPs, indicatingits potential as a novel and cost-effective expression systemfor subunit vaccines. However, the capacity and associatedproduction levels of A. niger for the assembly of complex

Appl Microbiol Biotechnol (2007) 75:279–288DOI 10.1007/s00253-006-0812-9

E. R. James : J. F. Görgens (*)Department of Process Engineering, University of Stellenbosch,Private Bag X1,7602 Stellenbosch, South Africae-mail: [email protected]

W. H. van ZylDepartment of Microbiology, University of Stellenbosch,Private Bag X1,7602 Stellenbosch, South Africa

immunogenic viral proteins into VLPs have not beenquantified. In the present study, the production levels ofhepatitis B surface antigen (HbsAg), by recombinant A.niger under optimized conditions in batch cultivation, werequantified for comparison to established yeast expressionsystems.

Optimization of major bioprocess parameters during thesubmerged cultivation of A. niger is essential for thequantification of the VLP production levels. In particular,the effect of both agitation and dissolved oxygen (dO2)concentration on fungal morphology, biomass yield andVLP production during micro-pelleted growth in batchculture were considered. Agitation, oxygenation and dO2

are closely related bioprocess parameters that can increasethe supply of nutrients and O2 to fungal cells. However, toohigh levels of agitation can increase shear forces, possiblyinducing cell stress and morphological changes due todamaged cell structures. High dO2 concentrations may leadto elevated intracellular levels of O2

− anions causing cellularoxygen toxicity (Bai et al. 2004). As a consequence, thesevariations in agitation intensity and dO2 concentration maypositively or negatively influence the physiological state ofthe microorganism, potentially leading to an impact uponcell growth, morphology, protein secretion and productformation. The independent optimization of agitation anddO2 levels under well-controlled conditions in a bioreactor isrequired to study the complex relationships between fungalbiomass, total protein and VLP production. As a result, thecapacity of A. niger to produce and assemble compleximmunogenic viral proteins into VLPs in comparison toestablished systems can be evaluated.

Materials and methods

Recombinant fungal strain

The recombinant microorganism used in this study was theuridine auxotrophic (pyrG), protease-deficient (prtT), non-acidifying (phmA) mutant A. niger DAHB11#8 strain(Gordon et al. 2000; Plüddemann and van Zyl 2003). Thisstrain carries the structural gene encoding the major viralenvelope protein of the hepatitis B virus under thetranscriptional control of the constitutive A. nidulansglyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter(Plüddemann and van Zyl 2003). The host strain A. nigerD15 was generously donated by Prof. van den Hondel(TNO, Zeist, The Netherlands).

Cultivation media

The recombinant A. niger strain was cultured in Aspergillusminimal growth medium without uridine (Plüddemann and

van Zyl 2003) containing 3.5% glucose and supplementedwith 0.1% casamino acids (Difco) and 0.5% yeast extract.

Innoculum preparation

A. niger spores were obtained from a densely conidiatingculture grown on rich agar medium for 96 h (Plüddemannand van Zyl 2003). The spores were collected within asterile physiological saline solution (NaCl, 0.9% w/v)and pre-grown in a shake flask cultivation amounting to10% of the bioreactor culture’s total working volume(800 ml). Enough spores were transferred to the precultureto ensure an innoculum concentration of 1×106 spores ml−1

in the bioreactor. The preculture was then incubatedon a rotary shaker (New Brunswick Scientific, Edison,WJ, USA) at 30°C and 120 rpm for 12 h. This strategyensured reproducible growth rates and morphologicalproperties, nullifying any effects caused by spore age.

Cultivation conditions

Two 10-l bioreactors (BioFlo 110 Non-Jacketed Vessels,New Brunswick Scientific (NBS), USA) with a workingvolume of 8 l and a single 30×20 mm 3-bladed down-flowpitched blade impeller were used for cultivation. Foraeration, sterile-filtered air (0.2 μm absolute filters) enteredthe bioreactor at 0.5 vvm. NBS gas mixers ensured directdO2 concentration control with a mixture of O2, air and N2

gas. The dO2 concentrations were analysed by an onlineNBS polarographic electrode, an online NBS pH electrodemeasured the acidity of the culture broth and the temper-ature was controlled at a constant 30°C (±0.1°C) by aheating blanket and a cooling water stream (0.7 l min−1)that passed through a stainless steel heat transfer coil. Theexhaust gases left the top of the reactor through a condensercooled with water circulating at −4°C (1.1 l min−1). In theexhaust gas of bioreactor cultures, the concentration ofO2 and CO2 were determined by photoacoustic gasanalysis systems (INNOVA AirTech Instruments 1309 GasAnalyser). Aeration rate and off-gas analysis data wereused to calculate the specific oxygen uptake rate (SOUR).Foam was suppressed by the addition of antifoamreagent (antifoam 289, Sigma-Aldrich), with the aid of alevel sensor. The culture pH, when it was controlled,was performed with the automatic addition of 2 M KOH/H2SO4.

Sample preparation

Samples were collected every 6–12 h. Mycelia wereseparated from the sample liquid broth by filtering thesample through sterile Miracloth (Calbiochem, USA, Cat.Nr. 475855) and squeezed well to remove excess liquid.

280 Appl Microbiol Biotechnol (2007) 75:279–288

Mycelia and culture supernatant were collected, snap frozenin liquid nitrogen and stored at −80°C.

Assay preparation

Mycelia samples were ground in a mortar and pestle underliquid nitrogen to a fine powder, and an aliquot of 0.3 gmycelia powder was placed into a 2-ml microcentrifuge tubeand stored at −80°C. Ground samples were suspended inprotein extraction buffer; one with Triton X-100 for theextraction of both the intracellular membrane- and cytoplas-mic-associated proteins and one without the Triton X-100 forthe extraction of the cytoplasmic fraction. The buffercomposition included: 50 mM NaP (pH 7), 1 mM ethyl-enediaminetetraacetic acid (EDTA), 20 μM phenylmethylsul-fonyl fluoride (PMSF) and 0.1% Triton X-100. Extracellularprotein determination was performed by concentratingsupernatant samples using size exclusion membranes in the50-ml Amicon® 8050 (GRACE company, USA).

Dry weight

Dry weight was determined gravimetrically using nitrocel-lulose filters (0.45 μm, Gelman Sciences). The filters werepredried in a microwave oven for 10 min and weighed. Aknown volume of culture was filtered and washed with anequal volume of double-distilled water. The filters were driedin an oven at 60°C for 120 h, allowed to cool and weighed.Dry cell weight was determined in triplicate and divided bybroth sample volume to give biomass concentration asgDW l�1

culture.

HBsAg concentration determination

The ARCHITECT HBsAg assay (Abbott laboratories,Wiesbaden, Germany) was used for the quantitative deter-mination of HBsAg, using HBsAg derived from humanserum as a standard. This assay is a ChemiluminescentMicoparticle Immunoassay (CMIA), which uses micropar-ticles coated with monoclonal anti-HBs to detect conforma-tional fully assembled epitopes of the HBsAg and did notdetect free and unassembled HBsAg proteins.

Total protein concentration determination

Intracellular total protein was determined using theBradford method (Bradford 1976) with bovine serumalbumin (BSA) as a standard.

Glucose concentration determination

Glucose determination was quantified by high performanceliquid chromatography (HPLC) analysis using an AminexHXP-87H column (BioRad) for separation (at 25°C) andultraviolet (UV) and refractive index detector (Techlab,Germany) for detection. Sulfuric acid (0.01 N) was used asthe mobile phase (flow rate = 0.5 ml min−1).

Calculation of specific oxygen uptake rate (SOUR)

The specific oxygen uptake rate (SOUR) was calculatedaccording to the stationary method as described by Pateland Thibault (2004).

Experimental design

Response surface methodology (RSM) was used to optimizethe fermentation parameters for enhancing HBsAg concen-tration. A central composite rotatable design (CCRD)composed from the superimposition of a two-level factorialdesign for two factors with replicates at the centre point andstar points was used in this investigation (CCRD; Box et al.1978; Box and Draper 1959). Table 1 shows the CCRD usedto study the interactive response pattern between agitationrate (100–600 rpm) and dO2 concentration (2–95%) anddetermine their optimum combination, each at five levels:−∞, −1, 0, 1 and ∞. xi is the dimensionless value of anindependent variable, and Xi is the real value of thatindependent variable. The CCRD shown in Table 2 wasarranged to fit an appropriate multiple regression model.Three replicates at the centre of design were used to estimatethe coefficient of variance. A second-order polynomialequation was used to fit the experimental data. The modelproposed for the response was:

Y ¼ b0 þX

bixi þX

biix2i þ

Xbijxixj ð1Þ

Table 1 Variables and their levels for central composite rotatable design (CCRD)

Variable Symbols Levels

Coded Uncoded −∞ −1 0 1 ∞

Agitation intensity (rpm) X1 x1 100 250 350 450 600dO2 concentration (%) X2 x2 2 20 50 80 95

x1 ¼ X1 � 350ð Þ=100 and x2 ¼ X2 � 50ð Þ=30

Appl Microbiol Biotechnol (2007) 75:279–288 281

Where Y represents the predicted response of totalintracellular membrane-associated HBsAg production perliter culture, and β0, βi, βii and βij are the coefficients ofinterception, linear, quadratic and cross-product terms,respectively. The quality of the observed data and theaccuracy of the model were verified by conducting arandomised experiment within the boundary conditions ofthe experimental design in triplicate at 350 rpm and 25%dO2 concentration. The fitted equation was expressed as acontour plot by using STATISTICA software version 7.1(StatSoft (2005) http://www.statsoft.com) to visualise therelation between the response and experimental levels ofeach factor and to deduce the optimal condition.

Results

HBsAg production by A. niger strain DAHB11#8 wascarried out in a batch lab scale stirred bioreactor undercontrolled conditions. Response surface methodology (RSM)and a two-factor-two-level central composite rotatable design(CCRD) were employed to evaluate the interactive responsepattern between agitation intensity and dO2 concentrationand their optimum combination.

Experimental design and statistical analysis

The response surface model predicting the intracellularhepatitis B VLP concentration in terms of the independentvariables x1 (agitation intensity) and x2 (dO2 concentration)was obtained with the aid of STATISTICA as Eq. 2 andgraphically illustrated in Fig. 1.

Y ¼ 959�450x1�177x2þ134x21 � 126x22 þ 210x1x2 ð2Þ

The high value of the coefficient of determination (R2)explains 97% of the total variation, while the coefficient ofvariation (CV=3.4%) indicates the high quality of data. AnF-value of 53.88 and a p value less than 0.01 suggest that theregression is statistically significant (data not shown). Thetests on the individual coefficients suggest that the quadraticx21 (p<0.005) and the linear x1 (p<0.05) terms of agitationrate, are statistically significant. The accuracy of the modelwas verified by conducting a randomised experiment withinthe boundary of the experimental design in triplicate at

Intra

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Fig. 1 Central composite rotatable design of agitation intensity (rpm)and dissolved oxygen concentration (dO2%) in a batch culture. Theinterdependent effects of agitation intensity and dissolved oxygenconcentration (dO2%) in a batch culture on maximum intracellularmembrane-associated HBsAg concentration during cultivation. Theresponse surface plot was performed in STATISTICA as 3D contourplots. Points have an associated error ±10% of the value of the point

Table 2 Experimental design and results of the CCRD

Experiment number Independent variables Repetitions Intracellular membrane HBsAg production

X1 X2

Agitation (rpm) dO2 conc. (%) Experimental Predicted Residual

μgHBsAg l�1culture

� �

1 350 50 3 959.780 958.646 1.1342 100 50 2 3,002.104 2,921.901 80.2033 600 50 2 597.514 670.063 −72.5494 350 95 2 577.273 409.115 168.1575 350 2 2 789.507 918.615 −129.1086 250 80 1 807.189 1,029.321 −222.1327 250 20 1 1,811.428 1,804.155 7.2738 450 80 1 518.268 549.460 −31.929 450 20 1 680.758 482.545 198.21310a 350 25 3 1,565.347 1,020.00 545.347

aExperimental run conducted to verify the accuracy of response surface model.


http://www.statsoft.com

350 rpm and 25% dO2 concentration. The experimentalintracellular HBsAg concentration was compared to themodels predicted response, with a prediction within 65% ofthe experimental value (Table 2). The maximum experimen-tal HBsAg production conditions are observed to lie at100 rpm and 50% dO2 concentration. However, the modelpredicts that with an agitation rate of 100 rpm and the furtherdecrease in dO2 concentration to 25% (at an accurateextrapolatable distance from the boundary of the experimen-tal design), intracellular HBsAg production will be at anoptimum 3:4 mg l�1

culture

� �. The predicted increase in intra-

cellular membrane-associated HBsAg production levels3:4 vs: 3mg l�1

culture

� �falls within the experimental standard

deviation (11%) and could therefore not be verifiedexperimentally based on the Student’s t test (p>0.1).

Effects of agitation intensity

Cultures grown at 100 rpm and 50% dO2 produced largefluffy pellets, indicating the growth of hyphal structures, incomparison to the small smooth pellets produced in culturesgrown at 350 rpm and 50% dO2. Filamentous clumpsformed in cultures grown at 600 rpm and 50% dO2 (datanot shown). Highest biomass concentrations of 12:9 gDWl�1culture (60 h) and 12:8 gDW l�1

culture (42 h) were obtained atagitation rates of 100 and 600 rpm, respectively (Fig. 2a).Maximum specific growth rate data correlated with the

specific glucose consumption rate g g�1DWh

�1� �

and maximumSOUR (mmol O2 l−1 h−1), which were highest in culturesgrown at 600 rpm, followed by cultures grown at 100 rpm(Table 3).

Hepatitis B VLP production was localised intracellularly,and required long culturing times of up to 100 h to fullyassemble (Fig. 2b). The intracellular HBsAg specificproduction rate, mg g�1

DWh�1

� �, increased with an increase

in agitation intensity (Table 3), while volumetric HBsAgproduction increased with the decrease in agitation intensity(Fig. 2b). In all cultures, the majority of intracellularHBsAg production (98%) was associated with the mem-brane fraction (data not shown). However, cultures grownat 100 rpm demonstrated significant HBsAg accumulationin both the intracellular membrane 3:0 mg l�1

culture

� �and

cytoplasmic fraction 590 μg l�1culture

� �(Fig. 3). With culture

age, HBsAg concentration in these cellular fractionsdecreased, coinciding with the extracellular release ofHBsAg VLPs into the culture supernatant (Fig. 3). Thehighest level of extracellular HBsAg 970 μg l�1

culture

� �was

observed at 160 h. A maximum total volumetric HBsAgproduct of 3:6 mg l�1

culture was recorded after 82 h (Figs. 2band 3). Thereafter, an accumulative loss in volumetricproduct was observed. Whether this loss of product is dueto HBsAg instability caused by proteolytic degradation and/or autolysis as a result of culture age or nutrient limitationsis uncertain. However, the percentage cellular proteins

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Fig. 2 Effect of agitation (rpm)in a batch culture on a biomassdry mass concentration, b intra-cellular membrane-associatedHBsAg production,c culture pH and d percentagecellular protein that HBsAgrepresents. Shown are data forempty circles, 100 rpm; emptysquares, 350 rpm; emptytriangles, 600 rpm controlled ata dO2 concentration of 50%.Results are based over acultivation time of 150 h


consisting of HBsAg (0.5 to 1.1%) in all cultivationsincreased steadily with time (Fig. 2d), emphasising therobustness and stability of the recombinant hepatitis BVLPs to potential proteolytic degradation in comparison tocellular proteins. The control of culture pH is notrecommended for the protease-deficient (prtT), nonacidify-ing (phmA) A. niger DAHB 11#8 strain, as pH-controlledcultivation studies showed a decrease in HBsAg productionlevels of 35% at pH 6 and 75% at pH 7 in cultures grown at350 rpm and 50% dO2. (data not shown). These findingswere attributed to a slimy mucilage layer on the pelletsurface at higher pH values observed during filtration ofmycelia (data not shown). During uncontrolled pH biore-actor studies, at agitation rates of 100 and 350 rpm at 50%dO2, the culture pH decreased significantly during earlycultivation before stabilizing at pH 7.4 after 80 h. Incontrast, culture pH at high agitation intensities (600 rpm)did not stabilize and continually decreased throughout thecultivation, coinciding with significantly lower HBsAglevels (Fig. 2c).

Effects of dO2 concentration

An increase in dO2 concentration from 2% (oxygen-limitedconditions) to 25% at 350 rpm resulted in a morphological

shift from filamentous growth (clumps) to large fluffy pellets(data not shown). Pellet fluffiness became less pronouncedindicating the repression of hyphal growth by an increase indO2 to 50 and 95% at 350 rpm, while pellet size decreased.The highest biomass production levels of 11:9 gDWl

�1culture

(101 h) and 11:1 gDWl�1culture (72 h) were obtained in oxygen-

limited conditions (2% dO2 concentration) and intermediateoxygen levels (25% dO2 concentration), respectively(Fig. 4a). The maximum specific growth rate (h−1) andspecific glucose consumption rate g g�1

DWh�1

� �increased

with an increase in dO2 concentration, while the maximumSOUR (mmol O2 l

−1 h−1) of the microorganism obtained thehighest levels of 6.45 mmol O2 l

−1 h−1 with cultures grownat 25% dO2 concentration (Table 4).

Hepatitis B VLP production was most efficient in culturesgrown at intermediate oxygen levels (25% dO2), withoptimum intracellular HBsAg levels of 1; 565 μgl�1

culture

(72 h) and production rate of 0:116 μg g�1DWh

�1 (Fig. 4b;Table 4). Hepatitis B VLPs were exclusively associated withthe intracellular membranes of the fungal cells (99%; datanot shown). Similar to the results obtained in Fig. 2d, thepercentage of total cellular protein that HBsAg productionrepresented (1 to 0.65%) increased steadily with time in allcultivations (Fig. 4d). At dO2 concentrations of 2 and 25%,culture pH remained constant throughout the cultivation

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Fig. 3 Volumetric HBsAg pro-duction: total, filled circles;intracellular membrane fraction,empty circles; intracellularcytoplasmic fraction, emptysquares; extracellularsupernatant, empty triangles andbiomass dry mass concentration,inverted filled triangles.Data was collected underbioreactor conditions of 100 rpmand 50% dO2 concentration

Parameters Agitation intensities (rpm)

100 350 600

Biomass specific growth rate μ (h−1) 0.051 0.043 0.062Intracellular HBsAg specific production rate PP/X μg � g�1

DW h�1� �

0.084 0.104 0.123Specific glucose consumption rate QS/X g g�1

DW h�1� �

0.147 0.127 0.214Maximum specific OUR (mmol O2 l

−1 h−1) 6.41 5.44 7.13

Table 3 Calculated yields andspecific rate parameters fromthe different agitation intensitiescontrolled at a dO2

concentration of 50%


without pH control, while at 50 and 95% dO2, the culture pHdecreased significantly during early cultivation beforestabilizing above pH 7 after 100 h (Fig. 4c).

Discussion

The potential of A. niger as a production system for subunitvaccines was assessed by optimizing the production ofVLPs in batch culture under well-controlled conditions. Inparticular, the optimization of agitation and dO2 concentra-tion for HBsAg production with the use of RSM wasaddressed. The interdependent effects of agitation and dO2

concentration affected both biomass and VLP production,with agitation having a significantly more dominant effect(Fig. 1). These physiological effects were attributed to themorphological adaptations by the microorganism due tochanges in the environmental conditions, thus, emphasizingcell morphology as a critical bioprocessing parameter for

the submerged cultivation of filamentous fungi (Znidarsicand Pavko 2001).

Although high agitation intensities (600 rpm at 50% dO2)enhanced metabolic activity (Papagianni et al. 1999; Paulet al. 1999) and biomass yield (Fig. 2a; König et al. 1981),heterologous (HBsAg) production was influenced negative-ly (Fig. 2b; Li et al. 2002; Smith et al. 1990; Ujcovaet al. 1980). The more highly branched filamentous hyphaeat higher agitation intensities (600 rpm) correlated withhigher shear forces (Papagianni et al. 1999; Smith et al.1990; Metz et al. 1981) and increased levels of cellularstress due to structural damage (Braun and Vecht-Lipshitz1991; van Suijdam and Metz 1981), thereby, negativelyeffecting hepatitis B VLP synthesis, assembly and stability.Culture pH was significantly affected at high agitation(600 rpm; Fig. 2c). Whether this is a result of theoverproduction of an undetermined culture acidifyingextracellular protease or compound is uncertain. This isalso the case regarding VLP product degradation. At low

Parameters dO2 concentration [%]

2 25 50 95Biomass specific growth rate μ (h-1) 0.021 0.040 0.043 0.049Intracellular HBsAg specific production rate PP/X μg� g�1

DW h�1� �

0.072 0.116 0.104 0.09Specific glucose consumption rate Q S/X g g�1

DW h�1� �

0.111 0.123 0.127 0.243Maximum specific OUR (mmol O2 l

−1 h−1) 4.02 6.45 5.44 4.12

Table 4 Calculated yields andspecific rate parameters fromthe different dO2 concentrationcontrolled at an agitationintensity of 350 rpm

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a bFig. 4 Effect of dissolved oxy-gen concentration (dO2%) in abatch culture on a biomass drymass concentration, b intracel-lular membrane-associatedHBsAg production,c culture pH and d percentagecellular protein that HBsAgrepresents. Shown are data forempty circles, 2 dO2%; emptysquares, 25 dO2%; emptytriangles, 50 dO2%; invertedempty triangles, 95 dO2%controlled at an agitationintensity of 350 rpm. Results arebased over a cultivation timeof 150 h


and intermediate agitation rates (100 and 350 rpm at 50%dO2), the microorganism adapted to micro-pelleted growth,accompanied by an increase in the size and roughness ofthe pellets (data not shown; Nielsen et al. 1995). Thebiomass concentration, maximum specific growth rate,HBsAg production, specific glucose consumption rate andSOUR were at their highest levels at 100 rpm incomparison to cultures grown at 350 rpm at a dO2

concentration of 50% (Table 3), suggesting that theshearing off of protruding hyphal structures at higheragitation rates has a negative effect on VLP synthesis.The optimum HBsAg production at a low agitation rate of100 rpm corresponded to the morphology of large, looselypacked, fluffy pellets.

Intermediate oxygenation (25% dO2 at 350 rpm) wascharacterised by the adhesion of mycelia and formation oflarge fluffy pellets (data not shown). These conditionsresulted in the highest HBsAg yields (Fig. 4b,d; Table 4),which coincided with a high biomass concentration andSOUR (Fig. 4a; Table 4). HBsAg production was repressedby either oxygen-limited (2% at 350 rpm) or oxygen-rich(95% at 350 rpm; Fig. 4b,d; Table 4) conditions. Theseresults were attributed to the microorganism’s ability tomorphologically adapt to the environmental changes in aneffort to stabilize cellular concentrations of O2. Underoxygen-limited conditions (2% dO2), the microorganismadopted a filamentous morphology that increased thesurface area per volume exposed to the environment in aneffort to increase the efficiency of oxygen uptake. Duringoxygen-rich conditions (95%) the formation of smallsmooth pellets was considered as a “protective strategy”against the increased levels of intracellular reactive oxygenspecies (ROS) availability, causing increased levels ofcellular oxidative stress (Bai et al. 2004). Under highculture dO2 concentrations, the studies of Bai et al. (2004)proposed that fungal cells generally adopt morphologicalforms, such as the adhesion of mycelia (Wongwicharn et al.1999), the formation of smaller and smoother pellets(Higashiyama et al. 1999; data not shown) and increasedpellet density (Cui et al. 1998). These strategies have thenet effect of protecting the inner cells of the pellet from thepotential ROS-mediated damage at high dO2 concentra-tions, reducing levels of intracellular oxidative stress, andthus, adopting a morphology that negatively affects HBsAgproduction.

In the recombinant A. niger system, the majority ofVLPs assembled and accumulated in the membranousorganelles, although under optimized conditions of lowshear (100 rpm) and intermediate dO2 concentration (50%),significant amounts of the HBsAgs accumulated in thecytoplasm and subsequently released into the supernatant.These optimized conditions also facilitated the highest

intracellular VLP levels, indicating an association betweenthe saturation of intracellular membranes with VLPs andtheir release into the cytoplasm and supernatant. Whethersuch secretion of VLPs into the supernatant is due to theoperation of the nonclassical secretion pathway (Cleves etal. 1996), or by cellular autolysis towards the end of thecultivation (Papagianni 2004), is not clear. However, suchextracellular production of heterologous protein could beadvantageous for purification procedures.

Experimental results at an agitation intensity of100 rpm and 50% dO2, obtained maximum total HBsAgproduction levels of 3:6 mg l�1

culture 350 μg g�1DW

� �at a

biomass concentration of 10:5 gDW l�1culture. The response

surface model predicted optimum cultivation conditions atan agitation rate of 100 rpm and 25% dO2, obtainingintracellular membrane levels of 3:4 mg l�1

culture. This isapproximately a tenfold increase in VLP production in abatch culture (including the possibility of VLP release intothe cytoplasm and supernatant) in comparison to HBsAglevels obtained by Plüddemann and van Zyl (2003) of0:4 mg l�1


� �at a biomass concentration of

2 gDW l�1culture in shake flask cultivation. These results

suggest that VLP production in A. niger is predominantlybiomass-associated.

The A. niger production system achieved productionlevels comparable to three commercially competing recom-binant microorganisms in batch cultivations. In batchcultivations, Saccharomyces cerevisiae achieved HBsAgyields of 2 mg l�1


� �(Gu et al. 1991), which

has been significantly improved by using a fed-batchprocess, resulting in HBsAg levels of 10� 20 mg l�1

culture

670 μg g�1DW

� �(Gu et al. 1991; Hsieh et al. 1988). Similar

results were obtained with Hansenula polymorpha: in batchcultures, HBsAg production reached levels of 1:6 mg l�1

culture

46 μg g�1DW

� �, while the implementation of fed-batch culti-

vation substantially increased HBsAg production levels to8� 9 mg l�1


� �(de Roubin et al. 1991). In

shake flask cultivation, recombinant Pichia pastoris obtainedlow HBsAg production levels 270 μg g�1

DW

� �while large-

scale fed-batch cultivation resulted in a significant increasein HBsAg production to 0:38 g l�1

culture 6:4 mg g�1DW

� �(Cregg

et al. 1987).In the aforementioned reports using yeast expression

systems, VLP production was quantified by means ofpolyclonal immunoassays, as opposed to the method ofmonoclonal immunoassays exclusively used in the currentstudy. However, comparing the HBsAg production levels ofthe present study to polyclonal-determined levels may notgive a fair indication of the potential of the Aspergillus VLPproduction system. Although polyclonal immunoassayshave long been used as a means in detecting antigens(Harlow and Lane 1988), this method has one major


disadvantage: the antibody response to a given antigentends to be broad, covering both specific and cross-reactiveepitopes. As a result, immunoassays based solely onpolyclonal antibodies may not be very specific to correctlyfolded and assembled VLPs (Andreotti et al. 2003) andresult in the overestimation of VLP production levels.Monoclonal immunoassays have improved the specificityof the assay by simplifying the selection of antibodiesdirected against chosen molecular targets and has thus beendeemed as a more reliable and accurate means to quantifythe production of correctly folded and assembled VLPs(Andreotti et al. 2003).

Optimization of the bioprocess parameters, agitationintensity and culture dO2 concentration, enhanced theproduction and assembly of complex immunogenic viralproteins into VLPs by the filamentous fungi, A. niger inbatch culture. The morphological type and physiology of A.niger were strongly dependent on the environmentalconditions created by these parameters, especially agitationintensity, with cultivation conditions amounting to the leastamount of cellular stress obtaining the highest intracellularhepatitis B VLP production levels. Thus, the A. nigerexpression system is attractive for further developmentbecause of high biomass-associated VLP production levelsin batch culture, the ability for extracellular release and thepotential for fed-batch cultivation. Future studies include acomprehensive fed-batch study controlled at optimumcultivation conditions (an agitation rate of 100 rpm anddO2 concentration of 25%), where the effects of variousfeeding strategies on hepatitis B VLP production in A. nigerwill be investigated.

Acknowledgment E. R. James was supported by the NationalResearch Foundation (NRF, South Africa).

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