PA

Aa

b

c

a

ARRAA

KA�FRR

1

i[cmcdrtctBlorbcdi

h1

Process Biochemistry 51 (2016) 1331–1337

Contents lists available at ScienceDirect

Process Biochemistry

jo ur nal home p age: www.elsev ier .com/ locate /procbio

roduction of �-glucosidase from wheat bran and glycerol byspergillus niger in stirred tank and rotating fibrous bed bioreactors

smaa Abdellaa,b, Tarek El-Sayed Mazeeda,c, Ashraf F. El-Bazb, Shang-Tian Yanga,∗

William G. Lowrie Department of Chemical & Biomolecular Engineering, The Ohio State University, Columbus, OH, USADepartment of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, EgyptChemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Giza, Egypt

r t i c l e i n f o

rticle history:eceived 16 May 2016eceived in revised form 15 June 2016ccepted 3 July 2016vailable online 4 July 2016

a b s t r a c t

Aspergillus niger NRRL 3112 can produce considerable amounts of �-1,4-glucosidase (BGL) when grownon wheat bran and glycerol as co-substrates. BGL production was first investigated in a stirred-tankbioreactor (STR) at 450 rpm and 2 vvm. About 5.4 U/mL BGL was obtained using spore suspension asinoculum, whereas a higher production of 9.3 U/mL was obtained using precultured cell pellets. Theproduction of BGL in batch, fed-batch and repeated batch modes in a rotating fibrous bed bioreactor (RFBB)

eywords:spergillus niger-glucosidaseed-batch fermentationepeated batchotating fibrous bed bioreactor

was then studied and compared to the STR. The highest BGL productivity of 1.78 U/mL/day was obtainedin the RFBB operated at a repeated batch mode, which was slightly higher than that (1.65 U/mL/day)obtained in the STR with preformed cell pellets and about 1.75-fold of that (1.02 U/mL/day) for the free-cell fermentation in STR inoculated with spores. This work demonstrated that the RFBB could provide anefficient process for �-glucosidase production from low-cost wheat bran and glycerol.

© 2016 Elsevier Ltd. All rights reserved.

. Introduction

The abundant and renewable lignocellulosic biomass is promis-ng feedstock for sustainable production of biofuels and chemicals1,2]. However, lignocellulosic materials usually must be firstonverted to fermentable sugars, mainly glucose, by enzy-atic hydrolysis involving synergistic actions of three different

ellulases: endo-�-1,4-glucanase (endoglucanase, EG), cellobiohy-rolase (CBH), and �-1,4-glucosidase (BGL) or cellobiase [3,4]. EGandomly cut the �-1,4-glucosidic linkages of cellulose to exposehe polysaccharide chain ends, which CBH acts upon to releaseellobiose and cellodextrins (cellotriose, cellotetraose, etc.). BGLhen hydrolyzes cellobiose and cellodextrins to produce glucose.GL increases the efficiency of cellulose hydrolysis by reducing cel-

obiose inhibition of CBH, which hinders the complete hydrolysisf cellulose [5–8]. Commercial cellulases produced by Trichodermaeesei and other filamentous fungi are highly potent in EG and CBH,ut low in BGL activity [9]. New enzyme cocktails developed forellulose hydrolysis are thus often supplemented with BGL pro-

uced by, for example, Aspergillus niger [9]. BGL also has many other

ndustrial applications [10], including uses in the hydrolysis of glu-

∗ Corresponding author.E-mail address: yang.15@osu.edu (S.-T. Yang).

ttp://dx.doi.org/10.1016/j.procbio.2016.07.004359-5113/© 2016 Elsevier Ltd. All rights reserved.

cosides of isoflavones for aglycones production in soy [11,12] andfor hydroxytyrosol release from olive byproduct [13].

There have been extensive efforts to find and develop suit-able microorganisms for BGL, and cellulases in general, productionthrough strain screening and engineering [3,9,10,14–16]. For eco-nomical production of enzymes, it is important to develop anefficient fermentation process with high product yields and desir-able activities from low-cost feedstocks [17–22]. Various fungalfermentation processes, including solid state fermentation and sub-merged fermentation in stirred tanks and air-lift bioreactors havebeen studied for cellulases production [9,23–26]. Current large-scale enzyme production is primarily carried out in conventionalstirred-tank reactors (STR), which have high shear stress caus-ing mycelial rupture and enzyme deactivation [27]. In addition,it is difficult to control the morphology of filamentous fungi inSTR [28], and undesirable fungal morphology could lead to poormass transfer and product secretion during fermentation [28–30].Cell immobilization on or in solid supports, including polyurethanefoam, alginate beads, loofah sponge, polyester fabrics, and cottontowel, has been widely studied and effectively used for fungal mor-phology control in submerged fermentation [31–35]. In particular,immobilization of fungal spores and mycelia in a rotating fibrous

bed bioreactor (RFBB) has been shown to have many advantages inculturing filamentous fungi for chemicals and enzymes production,including increased product titers, productivities, and operationalstability [34–37].

1 chem

poeoeicpAmhgf

2

2

vtaw3am3NTmsfia(o5f

2

wcgaAs

2

tAup1ntttoabme

332 A. Abdella et al. / Process Bio

The objective of this research was to develop an economicalrocess for BGL production from low-cost substrates. Our previ-us screening and medium optimization studies in batch culturexperiments have shown that A. niger NRRL 3122 was capablef producing large amounts of BGL with wheat bran and glyc-rol as substrates [38]. Wheat bran was a superior substrate andnducer for BGL production, while glycerol was used as a low-costo-substrate for its availability from the biodiesel industry. In theresent work, we further studied and compared BGL production by. niger in STR and RFBB operated at fed-batch and repeated batchodes. Our results showed that stable BGL production by A. niger at

igh titer and productivity could be achieved from wheat bran andlycerol in the RFBB, offering a potentially more efficient processor BGL production.

. Materials and methods

.1. Cultures and media

A. niger NRRL 3122, obtained from Agricultural Research Ser-ice (ARS) Culture Collection (Peoria, Illinois USA), was used inhis study. The stock culture was maintained on potato-dextrose-gar (PDA) slants at 4 ◦C and sub-cultured every 4 weeks. Sporesere harvested by scraping from PDA slants (7 days incubated at

0 ◦C) with 10 mL distilled water to give a spore suspension ofpproximately 2 × 107 spores/mL. Unless otherwise noted, the fer-entation medium contained (g/L): 5 glycerol (analytical grade),

.5 wheat bran, 7.5 corn steep liquor (CSL, 50% dry matters), 1aNO3, 0.3 K2HPO4, 0.1 KCl, MgSO4·7H2O, and 0.01 FeSO4·7H2O.his medium composition was chosen based on our previousedium optimization study [38]. Wheat bran, obtained from a local

upermarket and used un-altered, contained (wt%) ∼15 starch, ∼50ber (cellulose and hemicellulose), ∼16 protein, and some vitaminsnd minerals. CSL containing (wt%) crude protein and amino acids25–45), lactic acid (10–25), reducing sugars (2.5), and ash (17) wasbtained from Cargill (Eddyville, IA). All media were adjusted to pH.6 with 1 N NaOH and sterilized by autoclaving at 121 ◦C, 15 psigor 30 min.

.2. Preparation of cell pellets

Cell pellets for seeding submerged fermentation in stirred tanksere prepared by inoculating spores (3.6 × 108) in a shake-flask

ontaining 150 mL of the preculture medium, which contained onlylucose (10 g/L) as the carbon source (no glycerol and wheat bran)nd all other medium components as the fermentation medium.fter inoculation, the flask was incubated at 30 ◦C on a rotatoryhaker at 200 rpm for 48 h.

.3. Fermentation kinetics studies in stirred-tank bioreactor

Batch fermentation kinetics was first studied in a 3-L stirred-ank reactor (STR) containing 1.5 L of the fermentation medium.fter autoclaving at 121 ◦C for 30 min, the bioreactor was inoc-lated with 0.9 mL of spore suspension (4 × 108) or 150 mL ofreformed cell pellets and operated at 30 ◦C with aeration at–2 vvm and agitation at 450 rpm for 10 days, unless otherwiseoted. The medium pH was not controlled during the fermenta-ion. Silicone antifoam 204 (Sigma-Aldrich) was added as neededo control foaming. For fed-batch fermentation, the bioreactor ini-ially contained 1 L of the medium and was pulse-fed with 500 mLf the medium containing 1.5 g/L wheat bran and 7.5 g/L glycerol

t 120 h. For the repeated-batch culture, 66% of the fermentationroth was removed and replaced with the same volume of freshedium containing 3.5 g/L wheat bran and 7.5 g/L glycerol at the

nd (∼240 h) of each batch. Unless otherwise noted, samples were

istry 51 (2016) 1331–1337

taken once per day, centrifuged at 13000 rpm for 10 min, and storedat −20 ◦C for further analysis.

2.4. Fermentation kinetics studies in rotating fibrous bedbioreactor

The rotating fibrous bed bioreactor (RFBB) was made of a 3-Lstirred-tank bioreactor with a perforated stainless steel cylinderaffixed with polypropylene cloth for cell immobilization mountedon the impeller shaft in the bioreactor (Fig. 1). The bioreactor with1.5 L of the fermentation medium was autoclaved at 121 ◦C for30 min. After cooling, the bioreactor was inoculated with 17 mL ofspore suspension (2 × 107/mL) and then operated at 30 ◦C with aer-ation at 2 vvm and agitation initially at 90 rpm and then increasedto 150 rpm when wheat bran and mycelia had been adsorbed on thepolypropylene cloth. The medium pH was not controlled during thefermentation. For the fed-batch culture, 500 mL of the medium con-taining 1.5 g/L wheat bran and 7.5 g/L glycerol was pulse-fed whenenzyme production had leveled off at 288 h. For repeated batch cul-ture, 66% of the fermentation broth was removed and replaced withthe same volume of fresh medium containing 3.5 g/L wheat branand 7.5 g/L glycerol at the end of the batch fermentation (∼288 h)as indicated by the cessation of enzyme production.

2.5. Analytical methods

The activity of �-glucosidase was assayed by the methoddescribed elsewhere [39]. Briefly, 0.5 mL of the substrate (0.4%cellobiose in 0.05 M citrate phosphate buffer, pH 4.8) was addedto 0.5 mL of appropriately diluted enzyme solution (fermentationbroth filtrate) and incubated at 50 ◦C for 30 min. The reaction mix-ture was placed in boiling water for 5 min to stop the reaction andthen immediately cooled in an ice bath. Standard �-glucosidase(4 U/mg, Sigma) was used as positive control under the same assayconditions. One unit of �-glucosidase activity is defined as theamount of enzyme producing 1 �mol glucose per min from cel-lobiose. The glucose concentration in the mixture was determinedby using a glucose analyzer (YSI Biochemical Analyzer, YellowSpring, Ohio) or high performance liquid chromatograph (HPLC)with an Aminex HPX–87H column (BioRad) at 45 ◦C and 0.005 MH2SO4 as the mobile phase at 0.6 mL/min [40]. The total reducingsugar concentration was determined by the DNS method [41]. Theconcentration of soluble protein present in the fermentation brothwas assayed using the commercial protein assay kit (BioRad Labora-tories, USA) with bovine serum albumin as the standard. All assayswere performed in triplicate.

3. Results and discussion

3.1. Free cell fermentation in stirred-tank bioreactor

Batch fermentation kinetics was first studied in STR. Fig. 2 showsthe kinetics for batch fermentation inoculated with either spores orpreformed cell pellets. The pH was not controlled, and it decreasedfrom the initial value of ∼5.5 to ∼2.8 for the one inoculated withspores (Fig. 2A) and more rapidly to ∼1.6 with cell pellets (Fig. 2B).The pH then went back up to ∼3.2 in both cases. A similar trendwas observed with the total reducing sugars, which decreased fromthe initial value of ∼2.5 g/L to ∼0.22 g/L at 120 h and then increasedslightly to ∼0.46 g/L due to the continuous saccharification of wheatbran [34,38]. Meanwhile, protein and �-glucosidase production

started after 24 h and increased to reach the maximum values of0.76 mg/mL and 5.4 U/mL, respectively, for the one inoculated withspores (Fig. 2A) and 1.63 mg/mL and 9.3 U/mL, respectively, withcell pellets (Fig. 2B). The specific BGL enzyme activity was lower

A. Abdella et al. / Process Biochemistry 51 (2016) 1331–1337 1333

Fig. 1. The rotating fibrous bed bioreactor for fermentation to produce �-glucosidase by A. niger.

Fig. 2. Batch fermentation kinetics of A. niger in a stirred-tank bioreactor inoculatedw(

fiw(Htofifci

0

0.5

1

1.5

2

2.5

3

0

1

2

3

4

5

6

7

8

9

0 50 10 0 150 200 250

pHβ-glu cosidase RSprotein

pH, β

-glu

cosi

dase

(U/m

l)

Time (h)

Prot

ein

(g/L

), R

S (g

/L)

RFBB

reached the maximum levels of 1.35 mg/mL and 8.1 U/mL, respec-

ith spores at 450 rpm and 1 vvm (A) or preformed cell pellets at 450 rpm and 2 vvmB). RS: reducing sugar.

or the latter case (5.7 U/mg protein vs. 7.1 U/mg protein), indicat-ng more proteins and perhaps other enzymes (such as xylanase)

ere produced with the preformed pellets. It is noted that glycerol5 g/L) was quickly consumed in the first 48 h (data not shown).igher protein and enzyme production in the fermentation with

he preformed pellets could be attributed to better cell morphol-gy and improved mass transfer. Both preformed pellets and pelletsormed from spores directly inoculated to the fermentor had a sim-lar and relatively uniform diameter of ∼2 mm. However, for the

ermentation inoculated with free spores, most wheat bran parti-les were covered by a thick and dense layer of mycelia, resultingn poor mass transfer and incomplete degradation of wheat bran in

Fig. 3. Batch fermentation kinetics of A. niger in RFBB inoculated with spores at150 rpm and 2 vvm. RS: reducing sugar.

the fermentation. There were also large amounts of freely dispersedmycelia present in the fermentor inoculated with free spores, whichalso increased the broth viscosity and further hampered masstransfer and �-glucosidase production. The results confirmed theimportance of controlling cell morphology in filamentous fungalfermentation for enzyme production [27–30].

3.2. Immobilized cell fermentation in rotating fibrous bedbioreactor

To control cell morphology during fermentation, spores wereimmobilized, via adsorption, onto the polypropylene cloth to forma biofilm, after spore germination, in the RFBB (Fig. 1). At thebeginning the medium was turbid due to suspended wheat branand spores, but became clear after several days. There were nosuspended mycelia or wheat bran particles after 10 days of fermen-tation and all cells were attached to the fibrous matrix forminga homogeneous biofilm, which grew to a thickness of ∼1 cm in7–10 days. Fig. 3 shows the fermentation kinetics in the RFBB for14 days. Since the pH was not controlled, it gradually decreasedfrom 5.5 to ∼2.6 at 96 h and then back up to ∼4.2 at 265 h. Thetotal reducing sugars decreased from the initial 2.36 g/L to ∼0.24 g/Lat 192 h and then increased slightly to 0.35 g/L at 265 h. Mean-while, protein and �-glucosidase production started at ∼72 h and

tively, at 240 h. The specific BGL enzyme activity was 6.0 U/mgprotein, similar to those obtained in the STR. It is noted that thebiofilm was made of a relatively porous layer of mycelia, allow-

1334 A. Abdella et al. / Process Biochem

A

B

0

0.5

1

1.5

2

2.5

3

0

2

4

6

8

10

12

0 50 10 0 150 20 0 25 0 300

pHβ-glucosidase RSprotein

pH, β

-glu

csid

ase

(U/m

l)

Prot

ein

(g/L

), R

S (g

/L)

Time (h)

STR, Fed-batch

0.0

0.6

1.2

1.8

2.4

3.0

3.6

0

2

4

6

8

10

12

0 50 100 150 200 250 300 350

pHβ-glucosidase proteinRS

pH, β

-glu

cosi

dase

(U/m

l)

Pro

tein

(g/L

), R

S (g

/L)

Time (h)

RFBB, Fed-batch

Fu(

iUmaieis

3

pswifffaar91t0et

ig. 4. Fed-batch fermentation kinetics of A. niger in a stirred-tank bioreactor inoc-lated with cell pellets at 450 rpm and 2 vvm (A) and RFBB at 150 rpm and 2 vvmB). RS: reducing sugar.

ng efficient mass transfer, cell growth, and enzyme production.nlike the free cell fermentation, there was no freely dispersedycelia, mycelial clumps or wheat bran in the fermentation broth

fter 10 days. The RFBB with the biofilm operated under low viscos-ty and low shear environment allowed for efficient cell growth andnzyme production [29,34,37]. Compared to free-cell fermentationn STR inoculated with spores, which produced only 5.4 U/mL BGL,ignificantly higher enzyme production was obtained in the RFBB.

.3. Fed-batch fermentation

Fed-batch fermentation is commonly used to extend productionhase and increase the final product titer when the limiting sub-trate is toxic to cells or has a low solubility. Fed-batch fermentationas thus studied in STR and RFBB by adding fresh medium contain-

ng wheat bran and glycerol, which were the limiting substratesor enzyme production. Fig. 4A shows the fermentation profilesor 14 days in STR inoculated with cell pellets. The pH decreasedrom 5.5 to 2.4 at 48 h and then increased to ∼3.1 at 96 h. After theddition of fresh medium at 120 h, it increased to 3.7 but declinedgain to 1.7 at 142 h and then increased gradually to 3.4. The totaleducing sugars decreased from the initial 2.7 g/L to ∼0.35 g/L at6 h. After the addition of fresh medium at 120 h, it increased to.1 g/L but then decreased rapidly to 0.25–0.37 g/L. Meanwhile, pro-

ein and �-glucosidase production started after 24 h and reached.57 mg/mL and 3.7 U/mL, respectively, at 96 h. The specific BGLnzyme activity was 6.5 U/mg protein, which was slightly higherhan that obtained in batch fermentation. After the addition of fresh

istry 51 (2016) 1331–1337

medium at 120 h, protein and �-glucosidase production contin-ued to reach their maximum levels of 1.73 mg/mL and 11.3 U/mL,respectively, at 288 h.

Similar profiles were observed with the RFBB fermentation(Fig. 4B), although the total time was longer for 17 days because theadditional time required for spore germination. The pH decreasedfrom 5.5 to 2.7 and then back up to ∼4.4 at 264 h. After the additionof fresh medium at 288 h, the pH increased to 5.0 and then declinedagain to 3.7 before increasing gradually to 4.1. The total reducingsugars decreased from the initial 2.9 g/L to ∼0.28 g/L then increasedto 0.44 g/L. After the addition of fresh medium at 288 h, it increasedto 1.47 g/L and then decreased to 0.40 g/L. Meanwhile, protein andenzyme production started after 72 h and increased to 1.04 mg/mLand 8.1 U/mL, respectively, by 240 h. The specific BGL enzyme activ-ity was 8.2 U/mg protein, which was significantly higher than thatobtained in batch fermentation. After the addition of fresh mediaat 288 h, they continued to increase to reach their maximum levelsof 1.30 mg/mL and 10.6 U/mL, respectively, at 360 h. Compared tobatch fermentation, higher protein and �-glucosidase productionwas obtained in fed-batch fermentation in both STR and RFBB dueto the replenish of limiting substrates (wheat bran and glycerol)allowing continued cell growth and enzyme production. It shouldbe noted that fed-batch fermentation with the RFBB also gave amuch higher specific BGL activity per total protein produced, whichwould be desirable for enzyme recovery and application.

3.4. Repeated batch fermentation

Repeated batch fermentation with cell recycle has the advan-tage of higher reactor productivity due to increased cell density inthe subsequent batches. Fig. 5A shows the repeated batch fermen-tation in STR inoculated with cell pellets operated for 16 days. In thefirst batch, the pH decreased from 5.3 to 2.4 and then increased to∼3.9 at 216 h. Meanwhile, the total reducing sugars decreased fromthe initial 3.5 g/L to ∼0.21 g/L, and protein and �-glucosidase pro-duction reached 1.59 mg/mL and 8.6 U/mL, respectively. The secondbatch started at 240 h by replacing 66% of the fermentation brothwith fresh medium. The medium pH rapidly decreased from theinitial value of 4.7 to 2.9 and then increased gradually to 5.3 at380 h. Meanwhile, total reducing sugars decreased from 2.82 g/L to0.38–0.45 g/L, and protein and �-glucosidase production increasedfrom 0.45 mg/mL to 1.74 mg/mL and 2.6 U/mL to 10.8 U/mL, respec-tively, by 360 h. The specific BGL activity was 5.4 U/mg protein forthe first batch and 6.2 U/mg protein for the second batch, both wereslightly lower than that obtained in fed-batch fermentation.

Similarly, repeated batch fermentations were also studied inRFBB (Fig. 5B). In the first batch, the pH decreased from 5.5 to 2.6 andthen back up to ∼4.3 at 264 h, the total reducing sugars decreasedfrom the initial 2.73 g/L to ∼0.24 g/L, and protein and �-glucosidaseproduction reached 0.9 mg/mL and 8.2 U/mL, respectively, at 240 h.After starting the second batch at 288 h, the medium pH decreasedfrom 5.2 to 3.6 and then increased gradually to 4.3 at 408 h. Mean-while, total reducing sugars decreased from 2.04 g/L to 0.27 g/L, andprotein and �-glucosidase production increased from 0.32 mg/mLto 0.95 mg/mL and 2.48 U/mL to 9.6 U/mL, respectively, by 384 h.The specific BGL activity was 9.1 U/mg protein for the first batch and10.1 U/mg protein for the second batch, both were slightly higherthan that obtained in fed-batch fermentation.

3.5. Comparison of fermentation performance under differentmodes

Table 1 summarizes and compares the results obtained in STRand RFBB under various operating modes. It is clear that betterfermentation performance was attained in the immobilized-cellfermentation with either cell pellets in STR or biofilm in the RFBB

A. Abdella et al. / Process Biochemistry 51 (2016) 1331–1337 1335

Table 1Comparison of �-glucosidase production by Aspergillus niger in STR and RFBB operated in different modes.

Fermentation mode STR RFBB

BGL activity Productivity BGL activity Productivity

(U/mL) (U/mg) (U/mL/day) (U/mL) (U/mg) (U/mL/day)

BatchSpores 5.41 ± 0.04 7.1 ± 0.1 1.02 ± 0.01 8.1 ± 0.4 6.0 ± 0.3 1.27 ± 0.04Cell pellets 9.3 ± 0.4 5.7 ± 0.3 1.21 ± 0.05

Fed-batch11.3 ± 0.6 6.5 ± 0.3 1.27 ± 0.07 10.6 ± 0.5 8.2 ± 0.4 1.51 ± 0.25

Repeated batch1 st batch 8.6 ± 0.1 5.4 ± 0.1 1.22 ± 0.01 8.2 ± 0.6 9.1 ± 0.6 1.11 ± 0.082nd batch 10.8 ± 0.2 6.2 ± 0.1 1.65 ± 0.03 9.6 ± 0.4 10.1 ± 0.4 1.78 ± 0.08

Fermentations in STR were inoculated with preformed cell pellets and RFBB inoculated wmL of fermentation broth (U/mL) and per mg of protein present in the broth (U/mg). STR:

A

B

0

0.6

1.2

1.8

2.4

3

3.6

0

2

4

6

8

10

12

0 50 100 150 200 250 300 350

pHβ-glucosidase proeinRS

Prot

ein

(g/L

), R

S (g

/L)

pH, β

-glu

cosi

dase

(U/m

l)

Time (h)

STR, Repeated batch

0

0.5

1

1.5

2

2.5

3

0

2

4

6

8

10

12

0 50 100 15 0 200 250 300 350 400

pHβ-glucosidase RSprotein

Time (h)

pH, β

-glu

cosi

dase

(U/m

l)

Prot

ein

(g/L

), R

S (g

/L)

RFBB, Repeated batch

Fi(

aTcaffiviBb

ig. 5. Repeated batch fermentation kinetics of A. niger in a stirred-tank bioreactornoculated with cell pellets at 450 rpm and 2 vvm (A) and RFBB at 150 rpm and 2 vvmB). RS: reducing sugar.

s compared to the free-cell fermentation with spores as inoculum.he better performance can be attributed to the better controlledell morphology, resulting in better mixing and mass transfer favor-ble to cell growth and enzyme production [29,34]. Fed-batchermentation extended the production period to reach a highernal product titer, while repeated batch fermentation increased

olumetric productivity due to increased cell density and the elim-nation of lag phase in subsequent batches. Although comparableGL titer and productivity were obtained with cell pellets in STR andiofilm in the RFBB, the latter gave a much higher (>30%) specific

ith spores, unless otherwise indicated. BGL activity is expressed as enzyme unit per stirred-tank reactor; RFBB: rotating fibrous bed bioreactor.

BGL activity, which would be more favorable for purification andindustrial application. Furthermore, the RFBB was easier to oper-ate under the repeated batch mode since all cells were attachedto the rotating fibrous bed and the cell-free broth allowed for eas-ier medium replacement and sampling without clogging of sampleport and tubing by large mycelia clumps or pellets. Direct sporeinoculation to the RFBB also eliminated the seed culture prepara-tion step needed in preparing cell pellets for the STR fermentation.Further improvements in the final product titer and productivitycan be achieved with medium and process optimization.

3.6. Comparison to other studies

Many bacteria, yeasts and filamentous fungi can produce BGL[24,42–59]. Among them, filamentous fungi with GRAS (generallyregarded as safe) status, such as Aspergillus, are among the bestenzyme producers [24,44–46]. The results from this study also com-pare favorably with those reported in the literature (see Table 2).With cells immobilized in the RFBB operated under the repeatedbatch mode, a high BGL productivity of 1.78 U/mL/day was attained,which was higher than most of the reported values in the literature.Although higher BGL productivities of 2.5–6.9 U/mL/day have beenreported [20,23,44,58], they were obtained with different species orstrains, grown on different substrates, or under different fermenta-tion conditions. Furthermore, the higher BGL production obtainedin solid state cultures would be difficult to scale up for industrialapplication. It should be noted that most prior studies did not reportthe specific BGL activity obtained in submerged fermentation. Thespecific BGL activity of 8–10 U/mg protein obtained in the RFBB fer-mentation was comparable to those of the commercial BGL, rangingfrom <1 U/mg to >50 U/mg, depending on the type of the products.

It should also be noted that the types and amounts (activities)of BGL produced in fermentation could be greatly affected by thesubstrate. For example, only two BGL isoforms were expressed byA. niger NII-08121 when wheat bran or rice straw was used as thecarbon source, whereas four different isoforms of BGL were pro-duced with lactose or cellulose but at much lower BGL activitiesand almost no BGL activity was detected with glucose as the sub-strate [22]. Different forms of cellulose used in the culture alsoaffected BGL production by Fusarium oxysporum, with methyl cel-lulose giving the highest production (33.1 U/mL) as compared toAvicel (16.1 U/mL) and CMC (15.1 U/mL) [56]. In addition, differentpretreatments of the same biomass substrate could also result inlarge differences in the amounts and types of cellulase, including

BGL, produced by the microorganism as found with A. niger grownon sugarcane bagasse [23] and Penicillium janthinellum grown onrice straw [57]. In our previous study, we found wheat bran wasthe best substrate and inducer for BGL production in submerged

1336 A. Abdella et al. / Process Biochemistry 51 (2016) 1331–1337

Table 2Comparison of �-glucosidase production from various substrates in different modes of fermentation by Aspergillus and other fungal species.

Microorganism Substrate Mode and type of reactor �-Glucosidase (U/mL) Productivity (U/mL/day) Reference

T. viridi Sugarcane bagasse + wheat bran STR 0.26 0.016 [34]T. citrinoviride mutant Potato dextrose (glucose) Shake flask 8.2 1.65 [55]Penicillium simplicissimum Wheat bran, rice straw, bean cake powder STR 0.15 0.037 [47]Penicillium funiculosum Avicel STR 2.4 0.29 [48]Penicillium janthinellum Rice straw Shake flask 0.3–3.1 0.038–0.38 [57]Fusarium oxysporum Papyrus paper Shake flask 2.3 0.12 [49]A. phoenicis Wheat bran Shake flask 4.0 0.45 [50]A. oryzae 3T5B8 Sugarcane bagasse Shake flask SSF 5.0–7.4 1.9–2.2 1.7–2.8 0.6–0.7 [23]A. oryzae CBS 12559 Quercetin Shake flask 2.2 0.16 [51]A. niger KKS 1–2% Rice straw Air-lift reactor 4 0.4 [52]A. niger NCIM 1207 Xylan + Urea + Glycerol Shake flask 19 1.35 [46]A. niger NRRL 3112 Wheat bran + glycerol RFBB STR 9.6 10.8 1.78 1.65 This studyA. wentii 3% Cellulose STR 10 1.25 [53]A. wentii CM-cellulose STR 13.8 1.7 [45]A. heterornorphus 0.5% glucose STR 17.6 2.5 [44]Gongronella butleri Wheat bran SSF 21.5 5.4 [20]Lichtheimia ramosa Wheat bran SSF 27.4 6.9 [58]

SSF

A d state

fftsN

4

ntcitb�bdwbowitacp

A

s

R

[

[

[

[

[

[

[

[

[

[

[

[

[

Thermomucor indicae-seudaticae N31 Soybean meal

ll data were from submerged fermentation, except for those indicated as SSF (soli

ermentation by A. niger [38]. A similar finding was also reportedor BGL production by Gongronella butleri in solid state fermenta-ion (SSF) [20], although soybean meal was reported to be the bestubstrate for BGL production by Thermomucor indicae-seudaticae31 [59].

. Conclusions

In general, the cultivation of filamentous fungi, including A.iger, in submerged fermentation needs to be approached with cau-ion. As found in the present study, inoculation with preformedell pellets of proper size, instead of spores, could have majormpact on enzyme production in STR because of improved massransfer inside the size-controlled pellets, which could enhanceoth nutrients transport into cells and the release of produced-glucosidase into the medium. Similarly, immobilized cells asiofilm in the RFBB also enhanced mass transfer and enzyme pro-uction. It also facilitated medium exchange and sampling as thereere no freely suspended mycelia or cells in the fermentation

roth. Consequently, the highest productivity of 1.78 U/mL/day wasbtained in the RFBB operated at the repeated batch mode, whichas about 3-fold of that for the free-cell batch fermentation in STR

noculated with spores. In conclusion, stable �-glucosidase produc-ion at high titer, productivity, and specific enzyme activity could bechieved by culturing A. niger on wheat bran and glycerol as low-ost substrates in the RFBB, offering an efficient and economicalrocess for producing �-glucosidase for industrial applications.

cknowledgement

The work was in part supported by the Cultural Affairs and Mis-ion Department, Ministry of Higher Education, Egypt.

eferences

[1] J. Wang, X. Yang, C.C. Chen, S.T. Yang, Engineering clostridia for butanolproduction from biorenewable resources: from cells to process integration,Curr. Opin. Chem. Eng. 6 (2014) 43–54.

[2] V. Parisutham, T.H. Kim, S.K. Lee, Feasibilities of consolidated bioprocessingmicrobes: from pretreatment to biofuel production, Bioresour. Technol. 161(2014) 431–440.

[3] Y.H.P. Zhang, M.E. Himmel, J.R. Mielenz, Outlook for cellulase improvement:screening and selection strategies, Biotechnol. Adv. 24 (2006) 452–481.

[4] R.R. Singhania, A.K. Patel, R.K. Sukumaran, C. Larroche, A. Pandey, Role andsignificance of beta-glucosidases in the hydrolysis of cellulose for bioethanolproduction, Bioresour. Technol. 127 (2013) 500–507.

[5] H. Teugjas, P. Väljamäe, Selecting �-glucosidases to support cellulases incellulose saccharification, Biotechnol. Biofuels 6 (2013) 105.

2.4 0.6 [59]

fermentation).

[6] V. Rani, S. Mohanram, R. Tiwari, L. Nain, A. Arora, Beta-glucosidase: keyenzyme in determining efficiency of cellulase and biomass hydrolysis, J.Bioprocess Biotechnol. 5 (2014) 197.

[7] D.G. Borges, B. Baraldo Jr., C.S. Farinas, R.L.C. Giordano, P.W. Tardioli, Enhancedsaccharification of sugarcane bagasse using soluble cellulase supplementedwith immobilized �-glucosidase, Bioresour. Technol. 167 (2014) 206–213.

[8] L. Gao, F. Gao, X. Jiang, C. Zhang, D. Zhang, L. Wang, G. Wu, S. Chen,Biochemical characterization of a new �-glucosidase (Cel3E) from Penicilliumpiceum and its application in boosting lignocelluloses bioconversion andforming disaccharide inducers: new insights into the role of �-glucosidase,Process Biochem. 49 (2014) 768–774.

[9] R.R. Singhania, R.K. Sukumaran, A.K. Patel, C. Larroche, A. Pandey,Advancement and comparative profiles in the production technologies usingsolid-state and submerged fermentation for microbial cellulases, EnzymeMicrob. Technol. 46 (2010) 541–549.

10] Y. Bhatia, S. Mishra, V.S. Bisaria, Microbial �-glucosidases: cloning, properties,and applications, Crit. Rev. Biotechnol. 22 (2002) 375–407.

11] C.L. Handa, U.R. Couto, A.H. Vicensoti, S.R. Georgetti, E.I. Ida, Optimisation ofsoy flour fermentation parameters to produce �-glucosidase forbioconversion into aglycones, Food Chem. 152 (2014) 56–65.

12] K. Horii, T. Adachi, T. Matsuda, T. Tanaka, H. Sahara, S. Shibasaki, C. Ogino, Y.Hata, M. Ueda, A. Kondo, Improvement of isoflavone aglycones productionusing �–glucosidase secretory produced in recombinant Aspergillus oryzae, J.Mol. Catal. B: Enzym. 59 (2009) 297–301.

13] M. Hamza, S. Sayadi, High production of Aspergillus niger �-glucosidase atpilot-scale and application for hydroxytyrosol release from olive by-product,Int. J. Food Sci. Technol. 50 (2015) 1882–1890.

14] B.S. Montenecourt, D.E. Eveleigh, Selective screening methods for theisolation of high yielding cellulase mutants of T. reesei, Adv. Chem. Ser. 181(1979) 289–301.

15] A.L. Allen, C.D. Roche, Effects of strain and fermentation conditions onproduction of cellulase by T. reesei, Biotechnol. Bioeng. 33 (1989) 650–656.

16] M. Zoglowek, P.S. Lübeck, B.K. Ahring, M. Lübeck, Heterologous expression ofcellobiohydrolases in filamentous fungi −An update on the current challenges,achievements and perspectives, Process Biochem. 50 (2015) 211–220.

17] J.M. Almeida, V.A. Lima, P.C. Giloni-Lima, A. Knob, Canola meal as a novelsubstrate for �-glucosidase production by Trichoderma viride: application ofthe crude extract to biomass saccharification, Bioprocess Biosyst. Eng. 38(2015) 1889–1902.

18] M.G. Adsul, J.E. Ghuleb, R. Singhb, H. Shaikhb, K.B. Bastawdea, D.V. Gokhalea,A.J. Varmab, Polysaccharides from bagasse: applications in cellulase andxylanase production, Carbohydr. Polym. 57 (2004) 67–72.

19] R.S.R. Leite, H.F. Alves-Prado, H. Cabral, F.C. Pagnocca, E. Gomes, R. Da-Silva,Production and characteristics comparison of crude �-glucosidases producedby microorganisms Thermoascus aurantiacus e Aureobasidium pullulans inagricultural wastes, Enzyme Microb. Technol. 43 (2008) 391–395.

20] F.R. da Silva Santos, N.F.L. Garcia, M.F. da Paz, G.G. Fonseca, R.S.R. Leite,Production and characterization of �-glucosidase from Gongronella butleri bysolid-state fermentation, Afr. J. Biotechnol. 15 (2016) 633–641.

21] A. Sørensen, J.J. Andersen, B.K. Ahring, P.J. Teller, M. Lübeck, Screening ofcarbon sources for beta-glucosidase production by Aspergillus saccharolyticus,Int. Biodeterior. Biodegradation 93 (2014) 78–83.

22] R.R. Singhania, R.K. Sukumaran, K.P. Rajasree, A. Mathew, L. Gottumukkala, A.Pandey, Properties of a major �-glucosidase-BGL1 from Aspergillus niger

NII-08121 expressed differentially in response to carbon sources, ProcessBiochem. 46 (2011) 1521–1524.

chem

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

314–319.[59] J. de Cassia Pereira, R.S.R. Leite, H.F.A. do Prado, D.A.B. Martins, E. Gomes, R. da

Silva, Production and characterization of �-glucosidase obtained by thesolid-state cultivation of the thermophilic fungus Thermomucorindicae-seudaticae N31, Appl. Biochem. Biotechnol. 175 (2015) 723–732.

A. Abdella et al. / Process Bio

23] V.M. Vasconcellos, P.W. Tardioli, R.L.C. Giordano, C.S. Farinas, Productionefficiency versus thermostability of (hemi)cellulolytic enzymatic cocktailsfrom different cultivation systems, Process Biochem. 50 (2015) 1701–1709.

24] I. Persson, F. Tjerneld, B.H. Hagerdal, Fungal cellulolytic enzyme production. Areview, Process Biochem. 26 (1991) 65–74.

25] H. Esterbauer, W. Steiner, L. Labudova, A. Hermann, M. Hayn, Production ofTrichoderma cellulase in laboratory and pilot scale, Bioresour. Technol. 36(1991) 51–65.

26] D.A.J. Wase, W.J. McManamey, S. Raymahasay, A.K. Vaid, Comparisonsbetween cellulase production by Aspergillus fumigatus in agitated vessels andin an air-lift fermentor, Biotechnol. Bioeng. 27 (1985) 1166–1172.

27] F.C. Domingues, J.A. Queiorz, J.M.S. Calbral, L.P. Fonesca, The influence ofculture conditions on mycelial structure and cellulase production byTrichoderma reesi Rut C-30, Enzyme Microb. Technol. 26 (2000) 394–401.

28] K. Zhang, C. Yu, S.T. Yang, Effects of soybean meal hydrolysate as the nitrogensource on seed culture morphology and fumaric acid production by Rhizopusoryzae, Process Biochem. 50 (2015) 173–179.

29] M. Talabardon, S.T. Yang, Production of GFP and glucoamylase byrecombinant Aspergillus niger: effects of fermentation conditions on fungalmorphology and protein secretion, Biotechnol. Prog. 21 (2005) 1389–1400.

30] A. Ahamed, P. Vermette, Effect of culture medium composition onTrichoderma reesei’s morphology and cellulase production, Bioresour. Technol.100 (2009) 5979–5987.

31] H. Akihiro, C.O. James, A. Hideki, T. Hideo, Acetylation of loffa (lufa cylindrica)sponge as immobilization carrier for bioprocess involving cellulase, J. Biosci.Bioeng. 103 (2007) 311–317.

32] L.M. Xia, X.L. Shen, High-yield cellulase production by Trichoderma reeseiZU-02 on corn cob residue, Bioresour. Technol. 91 (2004) 259–262.

33] Z. Wang, Y. Wang, S.T. Yang, R. Wang, H. Ren, A novel honeycomb matrix forcell immobilization to enhance lactic acid production by Rhizopus oryzae,Bioresour. Technol. 101 (2010) 5557–5564.

34] T.Q. Lan, D. Wei, S.T. Yang, X. Liu, Enhanced cellulase production byTrichoderma viride in a rotating fibrous bed bioreactor, Bioresour. Technol.133 (2013) 175–182.

35] N. Thongchul, S.T. Yang, Controlling biofilm growth and lactic acid productionby Rhizopus oryzae in a rotating fibrous bed bioreactor: effects of dissolvedoxygen, rotational speed, and urea concentration, J. Chin. Inst. Chem. Eng. 37(2006) 49–61.

36] A. Tay, S.-T. Yang, Production of l(+)-lactic acid from glucose and starch byimmobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor,Biotechnol. Bioeng. 80 (2002) 1–12.

37] Z.-N. Xu, S.-T. Yang, Production of mycophenolic acid by Penecilliumbrevicompactum immobilized in a rotating fibrous-bed bioreactor, EnzymeMicrob. Technol. 40 (2007) 623–628.

38] A. Abdella, T.E.L. Mazeed, S.T. Yang, A.F. El-Baz, Production of �-glucosidaseby Aspergillus niger on wheat bran and glycerol in submerged culture:factorial experimental design and process optimization, Curr. Biotechnol. 3(2014) 197–206.

39] M. Zaldívar, J.C. Velásquez, I. Contreras, L.M. Pérez, Trichoderma aureoviride, amutant with enhanced production of lytic enzymes: its potential use in wastecellulose degradation and/or biocontrol, Electron. J. Biotechnol. 4 (2001) 1–9.

40] S. Suwannakham, S.T. Yang, Enhanced propionic acid fermentation byPropionibacterium acidipropionici mutant obtained by adaptation in afibrous-bed bioreactor, Biotechnol. Bioeng. 91 (2005) 325–337.

41] G.M. Miller, Use of dinitrosalicylic acid reagent for determination of reducingsugar, Anal. Chem. 31 (1959) 426–428.

42] C. Zhang, D. Li, H. Yu, B. Zhang, F. Jin, Purification and characterization ofpiceid-�-d-glucosidase from Aspergillus oryzae, Process Biochem. 42 (2007)83–88.

43] A.B. Dhake, M.B. Patil, Production of �-glucosidase by Penicilliumpurpurogenum, Braz. J. Microbiol. 63 (2005) 170–176.

istry 51 (2016) 1331–1337 1337

44] I.V. Solovyeva, V.M. Ananjin, A.V. Boev, O.N. Okunev, The controlledbiosynthesis of cellobiase by Aspergillus fungi, Process Biochem. 32 (1997)21–28.

45] V. Kandari, I. Vajpayee, D. Kumar, S. Gupta, Cellulase and �-glucosidaseproduction by Trichoderma viride and Aspergillus wentii in sub-mergedfermentation utilizing pretreated lignocellulosic biomass, J. Microbiol.Biotechnol. Res. 3 (2013) 63–78.

46] U. Khisti, K.B. Bastwade, D.V. Gokhale, Hyper production of �-glucosidase and�-xylosidase by Aspergillus niger NCIM 1207 in xylan containing media,Bioresources 6 (2011) 2066–2076.

47] H. Bai, H. Wang, J. Sun, M. Irfan, M. Han, Y. Huang, X. Han, Q. Yang, Production,purification and characterization of novel �-glucosidase from newly isolatedPenicillium simplicissimum H-11 in submerged fermentation, EXCLI J. 12(2013) 528–540.

48] M.L.A. Carvalho, D.F. Carvalho, E.B. Gomes, R.N. Maeda, L.M.M. Santa Anna,A.M. Castro, N. Pereira Jr., Optimisation of cellulase production by Penicilliumfuniculosum in a stirred tank bioreactor using multivariate response surfaceanalysis, Enzyme Res. (2014) 1–8, Article ID 703291.

49] S.A. Ismail, A.F. Sahab, S.S. Darwish, Factors affecting cellulase and�-glucosidase activities of Fusarium oxysporum isolated from old documents,Am. Eurasian J. Agric. Environ. Sci. 2 (2007) 689–695.

50] S. Jager, A. Braumber, E. Feher, K. Reczey, L. Kiss, Production andcharacterization of �-glucosidase from different Aspergillus strains, World J.Microb. Biotechnol. 17 (2001) 455–461.

51] C. Riou, J.M. Salmon, M.J. Vallier, Z. Gunata, P. Barre, Purification,characterization, and substrate specificity of a novel highly glucose-tolerant�-glucosidase from Aspergillus oryzae, Appl. Environ. Microbiol. 64 (1998)3607–3614.

52] S.W. Kim, S.W. Kang, J.S. Lee, Cellulase and xylanase production by Aspergillusniger KKS in various bioreactors, Bioresour. Technol. 59 (1997) 63–66.

53] S.K. Srivastava, K.B. Ramachandran, K.S. Gopalkrishnan, �-Glucosidaseproduction by Aspergillus wentii in stirred tank bioreactor, Biotechnol. Lett. 3(1981) 477–480.

54] X. Shen, L. Xia, Production and immobilization of cellobiase from Aspergillusniger ZU-07, Process Biochem. 39 (2004) 1363–1367.

55] M. Chandra, A. Kalra, N.S. Sangwan, S.S. Gaurav, M.P. Darokar, R.S. Sangwan,Development of a mutant of Trichoderma citrinoviride for enhancedproduction of cellulases, Bioresour. Technol. 100 (2009) 1659–1662.

56] F.M. Olajuyigbe, C.M. Nlekerem, O.A. Ogunyewo, Production andcharacterization of highly thermostable �-glucosidase during thebiodegradation of methyl cellulose by Fusarium oxysporum, Biochem. Res.Int. (2016) 1–8, Article ID 3978124.

57] B. Sharma, R. Agrawal, R.R. Singhania, A. Satlewal, A. Mathur, D. Tuli, M. Adsul,Untreated wheat straw: potential source for diverse cellulolytic enzymesecretion by Penicillium janthinellum EMS-UV-8 mutant, Bioresour. Technol.196 (2015) 518–524.

58] N.F.L. Garcia, F.R. da Silva Santos, F.A. Gonc alves, M.F. da Paz, G.G. Fonseca,R.S.R. Leite, Production of �-glucosidase on solid-state fermentation byLichtheimia ramosa in agroindustrial residues: characterization and catalyticproperties of the enzymatic extract, Electron. J. Biotechnol. 18 (2015)