Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

* Corresponding authors: e-mail: abdelazizamany@gmail.com

129

Induction of Glucose- Fructose Oxidoreductase Enzyme

Production Using Gamma- Irradiated Zymomonas Mobilis.

*A. B. Abd EI-Aziz and Eman Araby Microbiology Department, National Center for Radiation Research and Technology, Atomic Energy

Authority, Nasr City, Cairo, Egypt.

Received: 20/4/2013 Accepted: 1/6/2013

ABSTRACT

In the present study, from three distinctive production media were used, media

III was selected as it gave the highest production of bio-products under study. The

effect of different inoculum sizes (2.0, 4.0, 6.0, and 8.0g/l) on the production of the

selected media were studied and the results indicated that 2.0g/l was the best

inoculum size. The effects of important independent factors on the fermentation of

simultaneous production of glucose –fructose oxidoreductase (GFOR) enzyme,

sorbitol, and gluconic acid by Zymomonas mobilis grown on the selected media

components with different concentrations, time , pH, using a Plackett–Burman (P–B)

design has been studied. The results suggested that, yeast extract, peptone,

concentration of carbon and pH were affected positively on the fermentation process;

meanwhile, culture time was affected negatively on it. The irradiation dose level of 50

Gy was the best from different tested doses of gamma irradiation (20Gy-1.0 kGy) for

the fermentation process. The effect of different wastes acid hydrolyzates, which were

used as a sole carbon source or as a mixture with the basic synthetic media, on

irradiated Z. mobilis (50 Gy) has been determined. The hydrolyzed extract of carob

pod, orange and banana peels, showed a higher yield of bio-products compared with

rice straw hydrolyzed extract.

Keywords: Zymomonas mobilis, Sorbitol, gluconic acid, fermentation, glucose-fructose

oxidoreductase, wastes hydrolyzates, radiation.

INTRODUCTION

Gluconic acid (GA) and sorbitol are chemical commodities with many applications in food and

chemical industries. About 50,000-60,000 tones of GA are annually produced worldwide using

glucose (1). However, use of GA and its derivatives is limited in use because of high price (about US$

1.20-8.50/kg)(2).The global demand for sorbitol is mainly a function of its low calories and distinctive

blend of functional and surface active properties as a humectant, sweetener, bulking agent, stabilizer,

softener, emulsifier and etc. Low calories, diversified applications and growth in emerging markets are

expected to be the catalysts in driving the demand of sorbitol in the coming years. Owing to the above

mentioned properties, it was expected that the sorbitol volumes will be increased to reach 2.5 Million

MT by 2017(3). The current chemical process for sorbitol production is based on catalytic

hydrogenation of glucose making the process expensive (4).

However, other methods can be used to obtain sorbitol, such as the biotechnological process by

fermentation. The global market for fermentation products was increased from $15.9 billion in 2008 to

$22.4 billion by the end of 2013(5), a compound annual growth rate (CAGR) of 7.0%. Industrial

enzyme applications have the second-largest share of the market and are expected to grow at a CAGR

of 8.9%, from $3.2 billion in 2008 to $4.9 billion in 2013.

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

031

The biological process for sorbitol production utilizes a periplasmic enzyme, glucose–fructose

oxidoreductase (GFOR; EC 1.1.99.28) of Zymomonas mobilis, to convert fructose and glucose to

equimolar amounts of sorbitol and gluconic acid, respectively(6). Zymomonas mobilis, a Gram-negative

bacterium, the obligately fermentative bacterium found in association with plants containing high

concentrations of sugars in their saps and fruit juices (7) characterized by its higher sugar uptake, and

lower biomass production. This bacterium is one of the few facultative anaerobic bacteria that can

metabolize glucose and fructose via the Entner–Doudoroff (E–D) pathway, which is usually present in

aerobic microorganisms (8, 9).

The GFOR enzyme operates by a ping-pong mechanism, implying that the enzyme has only one

binding site, catalyzing the reaction of one of its substrates to yield a product that dissociates before

the other substrates bind. Hence, the overall reaction consists of two half reactions, with alternate

reduction of the bound NADP+ (as glucose is oxidized to gluconolactone) and oxidation of NADP (as

fructose is reduced to sorbitol).The gluconolactone is subsequently converted to gluconic acid;

however, sorbitol is not further metabolized by the cell (6).

GFOR seems to be specific for the acceptor molecule, fructose, but not for the donor aldose.

Consequently, several aldose sugars other than glucose such as xylose, galactose and even lactose (10)

can be oxidized into the corresponding aldonolactone, subsequently hydrolyzing into aldonic acid.

The agricultural wastes increasing, environmental and political pressures has increased

industrial focus toward alternative carbon and nitrogen sources. According to FAO (11), the total waste

generated from fruits was estimated as 3.36 million tones (MT) out of the total production of 16.8 MT.

Vegetables and some fruits yield between 25% and 30% of non-edible products (12). The citrus

processing industry yearly generates tons of residues, including peel and segment membranes. Banana

ranks second among fruit production after citrus, contributing to about 16% of the world’s total fruit

production. It is fourth on the list of the developing world’s most important food crops, after rice,

wheat and maize (12). Burning one tone of this wastes produce 1.7 tons of CO2, N2O, methyl chloride

and other poisoning gases. The wastes output had been increased to a point where natural reclamation

pathways are rendered inadequate. Alternative processes to give added value to this wasted material

must be considered (13). It is interesting to study a microorganism that could produce biological bio-

products from these wastes.

This study was carried to explore the optimum media composition for enhancement the

production of GFOR enzyme, sorbitol, and gluconic acid by Z. mobilis. Study the effect of low doses

of gamma radiation in the fermentation process, as well as to evaluate the effect of replace or

supplement the growth media with wastes hydrolyzates.

MATERIALS AND METHODS

Chemicals:

Fructose, sorbitol, glucose, Bovine Serum Albumin (BSA), p-nitrophenol and N-

morpholinoethanesulfonic acid (MES), were purchased from Sigma Chemical Co., St. Louis, MO,

USA. All other chemicals were of reagent grade and purchased from the local suppliers.

Bacterial strain and preservation:

Zymomonas mobilis ZM4 (ATCC 31821) was purchased from the American Type Culture

Collection (ATCC, Washington, DC, USA);via Cairo MERCIN, Faculty of Agriculture, Ain Shams

University, Cairo, Egypt.

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

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The Zymomonas mobilis was maintained in the liquid medium at 4°C and was reactivated every

30 days. Z. mobilis was cultivated for 24 hrs at 30°C, 130 rpm and at pH 5.5. pH was adjusted with

5M KOH solution. The Medium used for maintenance, and inocula production (preculture medium)

contained (g/L): sucrose 100; yeast extract 10; peptone 10; (NH4)2SO4 1; KH2 PO4 2; Mg SO4.7H2O

0.5. Sucrose was autoclaved separately at 115 °C for 15 min. The medium was sterilized at 121 °C for

20 min (14).

Selection of media composition:

Formulation of the different media, for growth and production of intracellular enzymes by

Zymomonas mobilis, was done according to Malvessi et al. (15); the culture media were composed of

carbon source, nitrogen source, and some micronutrients.

Zymomonas mobilis ZM4 (ATCC 31821) was grown at 30oC in three different media. The first

medium (Medium I) containing 200 g/L glucose, 1 g/L (NH4)2SO4, 1 g/L MgSO4.7H2O and 5 g/L

yeast extract (16), no phosphate salts were added in order to minimize the levels of phosphorylated

intermediates in the cells that are necessary for ethanol production (17). Medium II composition was,

200g/L sucrose, 2 g/L KH2PO4, 1 g/L (NH4)2SO4, 2 g/L MgSO4.7H2O, 1 g/L yeast extract and peptone

2g/L, and the composition of the last medium was, 200g/L (100 glucose +100 fructose), 2 g/L

KH2PO4, 1 g/L (NH4)2SO4, 5.0 g/L yeast extract, and 5.0 g/L peptone (medium III).

Fermentation process:

The experiments were carried out in 250 mL Erlenmeyer flasks with 50 mL of medium I, II, or

III at 30ºC, static culture, for 48 hrs with initial pH 5.5. The inoculum was standardized at 2 g/L

(based on dry weight) for all of these experiments. The process was monitored by quantifying gluconic

acid, sorbitol and GFOR enzyme production. The experiments were carried out in duplicate and the

results presented here correspond to the average of the values obtained.

Effect of inoculum size:

Different inoculums (2, 4, 6, and 8g/L) were added to 250 mL conical flasks each contained 50

mL of the optimal fermentation medium, determined from the previous experiments, at 30ºC, static

culture, for 48 hrs with initial pH 5.5.

Statistical analysis:

Factors in the growth medium affecting GFOR production (medium III) were evaluated by The

Placket-Burman design (18). The variables chosen for the present study were the carbon source, main

nitrogen source (yeast extract and peptone), besides three physical parameters, which were pH,

agitation speed (rpm), and incubation time (hrs).

The variables were coded as; initial sugar concentration (X1) which was equal amounts of

(glucose + fructose), pH (X2), shaking (X3), incubation time (X4), yeast extract (X5), and peptone

(X6). The factors were investigated at two levels of variation (Table 1).

Fermentation treatments according to Plackett–Burman (P–B) design (18).

Plackett-Burman experimental design is a partial factorial design; here large numbers of

independent variables (N=6) are studied in limited number of experiments (12). a 50 mL volume of

media, with pH and total sugar content as specified in Table 1, was pipetted into a 250-mL Erlenmeyer

flask. The specified amount of yeast extract and peptone were added to it. The mixture was sterilized

at 121 ºC for 15 min, and it was inoculated with suitable volume of Z. mobilis bacterial suspension to

provide 2.0 g/L bacterial dry weight. The culture was incubated at 30ºC and specified rpm and period

according to Table 1.

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

031

In Table 1, each row represents an experiment and each column represents different variables.

For each nutrient variable, two different concentrations high (+) and low (-) were tested (Table 1). The

dependent variables (response) were sorbitol, gluconic acid, and GFOR enzyme produced in culture

media.

Experimental Data Analysis:

Experimental data were analyzed by the standard methods of Plackett-Burman (18). The effect of

each variable was determined with the following equation:

NXi

LXi

HXiV /)(

Where, Vxi is the concentration effect of the tested variable; Hxi and Lxi are the concentration of

product at high level and low level of the same variable, and N is the number of trials. When the sign

is positive, the influence of variable upon product production is greater at high concentration, and

when the sign is negative, the influence of variable is greater at a low concentration.

Table (1): P–B design experiments to investigate the effects of six selected variables (X1, X2, X3, X4,

X5, and X6) on sorbitol, gluconic acid, and GFOR production by Z. mobilis ZM4 (ATCC

31821).

Run Sugar

X1

pH

X2

Shaking

X3

Time

X4

Yeast extract

X5

Peptone

X6

1 200 5.5 150 24 5 5

2 200 5.5 150 48 5 10

3 200 7.0 0.0 48 5 5

4 200 5.5 0.0 24 10 10

5 100 5.5 0.0 48 10 10

6 100 7.0 150 48 5 10

7 100 5.5 0.0 24 5 5

8 200 7.0 0.0 48 10 5

9 100 7.0 150 24 10 5

10 200 7.0 150 24 10 10

11 100 5.5 150 48 10 5

12 100 7.0 0.0 24 5 10

Effect of gamma- irradiation:

Irradiation process was carried out at National Center for Radiation Research and Technology,

Nasr City, Cairo, Egypt. Z. mobilis was subjected to the γ-irradiation by cobalt-60 gamma chamber

4000A. INDIA, The average dose rate of this gamma radiation source was 1 kGy/15 min at the time of

the experiments. Ten mL cell suspension was transferred in each vial, sealed with paraffin and

exposed to the gamma irradiator. Different doses of gamma radiation were selected (0, 20, 30, 40, 50,

Indications Factors Low

levels

(-)

High

levels

(+)

X1 Initial sugar, g/L 100 200

X2 PH 5.5 7.0

X3 Shaking rate per minute,

(rpm)

0 150

X4 Time, hrs 24 48

X5 Yeast extract, g/L 5 10

X6 Peptone, g/L 5 10

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

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75, and 150 Gy, 0.25, 0.50, and 1.0 kGy). The treated cell suspension (2.0g/L) after different time

intervals was transferred to fermentation medium which contained (g/L) sugar 200 (glucose 100 +

fructose 100); yeast extract 10; peptone 10; KH2PO4 2; (NH4)2SO4 1; pH 7.0 and incubated at 30oC for

24 hrs at 150 rpm (according to the results of the previous experiment).

Samples Collection:

Samples of rice straw used in this study were collected from Meet Gamr Farms. Carob pods

(after removing its seeds), Banana and Orange (take its peels) were from local markets.

PREPARATION OF SAMPLES

Size reduction:

Drying, grinding, and sieving were done after the collection of the samples. The maximum

particle sizes of the ground samples were 2 mm. From each sample, 3kg was used for preparation of

hydrolyzates.

Steam treatment (Hydro-thermolysis):

Every different sample was separately treated. Samples were autoclaved at 121°C for 30 min

using water to solid ratio of 10 mL/ 1g (v/w), then released the pressure and cooling. This process

conditions were selected to maintain a furfural concentration below 1g/L (19). After cooling the liquid

parts were separated from the solid parts (non soluble portion) by filtration and kept in the freezer until

use.

Acid Hydrolysis:

The insoluble portions were soaked for 24hrs. in 0.75% (v/v) diluted sulfuric acid. The samples

were then hydrolysis at 180 °C for 15 min. After hydrolysis neutralized with 10M NaOH until the pH

became around 7.0. Separate the solid particles from the liquid. The solid parts were washed with

distilled water twice, to extract all soluble sugars from the solid material. The liquid parts (from steam

treatment and acid hydrolysis) were then centrifuged at 4,000g for 15 min, and the supernatant (pH

7.0) was evaporated at 64–67°C to a concentrate of about 50%w sugar (glucose equivalent). The

concentrate was stored at 4°C to make solutions of different concentrations. The concentrate is more

resistant to spoiling than diluted one.

Fermentation of Hydrolyzate:

A suitable volume of each hydrolyzed with pH 7.0 and sugar content of 200 g/L (original liquid)

was separately pipetted into a 250-mL Erlenmeyer flask or mixed with other sugars (sucrose, glucose,

or fructose by the equal amount; 100g/L for each carbon source) and mixed with distilled water, to

become a 100-mL volume culture according to Table 7. The amounts of other media components were

added to the flasks according to the previous experiments (yeast extract 10g/L; peptone 10 g/L; g/L

KH2PO4 2 g/L; (NH4)2SO4 1 g/L; pH 7.0). The mixture was sterilized at 121°C for 15 min, and then

inoculated with 2.0 g/L of irradiated (50 Gy) Z. mobilis bacterial suspension (based on dry weight).

This culture was incubated at 30°C and 150 rpm for 24 hrs.

Analytical methods:

Reducing sugars (RS) were quantified by Somogy method (20). Total reducing sugars (TRS)

were determined after acid hydrolysis with 0.1N HCl according to Amorim et al. (21). Glucose was

used as the standard sugar. The biomass was determined as g/ L by measuring optical density (OD) at

600 nm and using a standard curve in the range 0.15–0.35 g/ L bacterial dry weight. Sorbitol

concentration was quantified by Sanchez,s method (22) and high performance liquid chromatography

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

031

(HPLC) using an aminex HPX 87C column (300mm X 7.8 nm), at 55ºC, with water as eluent at flow

rate 1 mL/min., and a refraction index detector.

Enzyme assay:

The GFOR activity was quantified using the same method as described by Zachariou and

Scopes (6) measuring the absorption change at 405 nm, at 25 °C and quantified by titration with HCl.

The routine spectrophotometric assay solution contained 0.4M glucose, 0.8M fructose, 10mM K–MES

buffer, pH 6.4 and 0.29 mM p-nitrophenol/ml. One unit of GFOR activity is defined as 1 μmol of

gluconic acid formed per minute at 25 ◦C. Alternatively, the activity was measured (5 ml total

volume) on a pH-Stat (glass electrode pH meter type with a Fisher pencil probe); titrating with 0.1 M

Tris base buffer to maintain the pH at 6.2. Protein determinations were done according to the dye

binding method of Bradford (23) using Bovine Serum Albumin (BSA) as standard.

RESULTS AND DISCUSSION

Effect of fermentation media composition:

As a preliminary step, the effect of substrate concentrations on the Z. mobilis GFOR producing

ability was investigated in three different media at the same temperature and pH values. In comparison

between the three tested media; increasing activities were observed with medium III, which contained

equal amounts of glucose and fructose (100g/L for each), as the highest GFOR total activity was

26.45U/L after 48 hrs. culture time. On the other hand, the over all productivity of medium II, which

contains 200g/L sucrose, was higher than medium I which contains 200g/L glucose (Table 2). Medium

III also was the best between the various media used for the fermentation process by Z. mobilis as

indicated by the maximum sorbitol production (48.18 g/L) and the highest gluconic acid productivity

(1.02 g/L. h.) yield. Therefore, Medium III was chosen for further experiments.

The conversion of glucose and fructose into gluconic acid and sorbitol was conducted by

Erzinger and Vitolo (24) in a batch reactor with Zymomonas mobilis cells. High yields (more than 90%)

of gluconic acid and sorbitol were attained at a substrate contained (glucose plus fructose at 1:1 ratio),

using cells with glucose-fructose-oxidoreductase activity of 75 U/L.

The bacterium Zymomonas mobilis presents potential for sorbitol production when grown in a

culture medium with high-sugar concentration. Sorbitol is produced and accumulated in the

periplasma of the bacterium to protect the cells from the harmful effects of high osmotic pressure that

results from the action of invertase on sucrose. The conversion of sucrose into glucose and fructose

increases the osmolarity of the medium (25).

The present data are in good agreement with that recorded by Márcio and Maria (25) who found

that increasing concentration of sucrose up to 300g/L led to an excessive elevation in the osmotic

pressure and may be decrease the GFOR and sorbitol production.

The results reported herein are in agreement with that recorded by Gilmar et al. (26) who reported

that, the highest specific and total activity of GFOR enzyme (12.6 U/g cells and 62 U/L) were

obtained with 153g/L (lower concentration of glucose) and higher substrate (209g/L) led to decreasing

activates (7.0 U/L) in fresh Z. mobilis cells. Meanwhile, Loos et al. (27) reported in their study that

when Z. mobilis grown in medium with access to both substrates (glucose plus fructose) fructose is not

primarily transported but can be utilized to form fructo-oligomers or levan, consequently decreasing

the GFOR and sorbitol production.

In the natural environment of Z. mobilis, only glucose and fructose would be present at a high

enough concentration to be acted on. Sorbitol production in culture is mainly confined to growth on

glucose-fructose mixtures (or sucrose), but small amounts have been detected when cells are grown on

glucose or fructose alone (25).

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

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Table (2): Effect of fermentation media composition on glucose –fructose oxidoreductase enzyme,

sorbitol and gluconic acid production.

Effect of inoculum size:

Calculated system productivity and products yield are from the most important characteristics of

the fermentation process. From the data obtained in Table 3, it may be observed that the yields of

fermentation process were not proportional to the inoculum size. The maximum biosynthesis of

products (GFOR total activity 26.85 U/L; sorbitol 48.92 g/L; and gluconic acid 49.73 g/L) was

observed at an inoculum size of 2.0g/L. Thus this inoculum size (2.0g/L) was employed for further

experiments. Such behavior is very common with Zymomonas mobilis free cells because the

production of metabolites is separated from cell growth (14).

Table (3): Effect of imoculum size on glucose –fructose oxiodoreductase enzyme, sorbitol and

gluconic acid production.

Legend as Table 2

Fermentation and optimization results:

In the current study, temperature was not included as one of the tested parameters affecting

GFOR production. Instead, all cultures were incubated at 30°C based on optimum temperature

suggested by other authors in previous studies (2, 25 and 28). The results of the feasibility test (Tables 2

and 3) showed that Z. mobilis was able to produce GFOR, sorbitol and gluconic acid in media III with

inoculum size of 2.0g/L. Therefore, studies were extended by performing Plackett–Burman design

experiments in two repetitions, as shown in Table 1. Data from the feasibility test and following

literature review aided in selecting temperature of 30°C and the low and high values of the six selected

variables.

The influence of the independent variables for this study such as sugar concentration, pH,

shaking, incubation time, yeast extract, and peptone, were studied in the responses: sorbitol, gluconic

acid, and GFOR enzyme using a Plackett–Burman (P–B) design Table 4. The results indicated that the

maximum GFOR activity obtained was 35.63U/L, the largest sorbitol (54.91g/ L), and gluconic acid (

Media no X

g/L

XV.P

g/L.h

AS.

U/g

AE.

U/L

G

g/L GV.P

g/L.h

I 27.00 0.56 5.49 14.82 24.69 0.51

II 32.36 0.67 6.46 17.77 38.49 0.80

III 48.18 1.01 14.07 26.45 48.46 1.02

X = Produced sorbitol (g/L).

V.PX =Sorbitol volumetric productivity (g/L.h).

S.A= Specific GFOR activity (U/g of cell).

E.A = Total activity (U/L).

G=Gluconic acid (g/L).

V.PG= Gluconic acid volumetric productivity (g/L.h).

inoculum

size

g/L

X

g/L

XV.P

g/L.h

AS.

U/g

AE.

U/L

G

g/L GV.P

g/L.h

2.0 48.92 1.02 8.44 26.85 49.73 1.046

4.0 46.18 1.92 13.04 25.35 47.50 0.99

6.0 45.09 1.88 14.18 24.75 46.27 0.96

8.0 42.18 0.88 5.74 23.15 44.95 0.94

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56.09 g/ L) production which were achieved in the 10th run with the specific conditions of initial

carbon source 200g/L, yeast extract 10g/L, peptone 10g/L, pH 7.0, shaking(150rpm), and incubation

period of 24hrs. While the minimum GFOR activity (17.27U/L), the smallest sorbitol (29.64g/ L), and

gluconic acid ( 30.94 g/ L) production was observed in run number 7, where all independent variables

at low levels (-).

Main effects of the examined variables on GFOR production were calculated when the sign of

the effect of the tested variable is positive, the influence of the variable on GFOR production is greater

at a high level and when negative, the effect of the variable is greater at a low level. Among the

variables screened, the most effective factors with high level were peptone (+3.299), rpm (+3.437),

and pH (+3.1975); variable yeast extract (+2.499) had the moderate positive effect; initial sugar

contents (+1.2785) had slight positive effects, whereas the variables time (-1.044) showed the negative

effect, according to the P–B responses presented in Table 1.

Our results are in accordant with that recorded by Márcio and Maria (25) who reported that

Z.mobilis cells growth was the highest at 24 hrs. but after this time the growth ceased. For ethanol

production from carob pod extract, Zymomonas mobilis PTCC 1718, was grown for 17 hrs at 30°C in

a conical flask shaken at 120 rpm in medium containing 10 g L-1 peptone , 10 g L-1 yeast extract, a

slight positive effect was observed in moving from pH 1.1 to 7.0 (29).

Zymomonas mobilis ATCC29191 was grown in fermentation medium containing (g/L): sucrose

200; yeast extract 10; peptone 5; supplemented with salts; at 30°C for 36 hrs. The inoculum was

standardized at 0.2 g/L (30). Z. mobilis (DSM 473) was cultivated anaerobically in an automatically

controlled 30 L fermenter fitted with a pH electrode for 36 hrs. at 30°C, 130 rpm and at pH 6.2. The

fermentation medium contained 200 g/L glucose, 5 g/L yeast extract, and salts (31).

Aziz et al. (31) reported that, there was a significant increase in cell production up to 24 hrs but

after 24 hrs. no increases in the cell concentrations were observed. Highest sorbitol production

occurred in the 36 hrs. culture period with a production of 38.09 g/L sorbitol, presenting a productivity

of 1.34 g. L/h that was not statistically different from the 24 hrs. time period. In the 48 hrs.

fermentation period, there was a significant drop in sorbitol production and the value of 20.22 g/L did

not differ statistically from that of 18 hrs. Boonyras et al. (32) cultivated Z. mobilis IFO 13756 in the

fermentation medium contained 10g/L yeast extract, 10g/L peptone, and 250g/L carbon source, at pH

7.5. Incubation was carried out at 30°C for 36 hrs. Z. mobilis grows best within the temperature range

of 30–35°C C and is can grow at pH values of 7.0-4.0 (33).

Table (4): Planning to investigate the effect of factors X1, X2, X3, X4, X5 and X6 on glucose –fructose

oxiodoreductase enzyme, sorbitol and gluconic acid production.

Run X

g/L

XV.P

g/L.h

AS.

U/g

AE.

U/L

G

g/L GV.P

g/L.h

1 40.00 1.67 3.40 21.96 42.36 1.77

2 42.00 0.88 5.29 23.07 43.68 0.91

3 32.55 0.68 2.48 17.87 31.89 0.66

4 44.73 1.86 5.48 24.55 45.57 1.90

5 35.10 0.73 3.83 19.26 36.25 0.76

6 49.10 1.02 6.67 26.95 50.61 1.05

7 29.64 1.24 3.87 16.27 30.94 1.29

8 45.64 0.95 8.52 25.05 45.77 0.95

9 45.27 1.89 5.58 24.85 46.23 1.93

10 54.91 2.29 13.36 35.63 56.09 2.34

11 41.45 0.86 5.35 22.76 42.82 0.89

12 43.82 1.83 6.159 24.53 44.40 1.80

Legend as Table 2

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

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Effect of gamma irradiation:

There is no report in the literature on the use of gamma- irradiation to stimulate sorbitol and

GFOR enzyme biosynthesis. However, the present investigation has attempted to modify the

productivity of Z. mobilis through exposing it to different irradiation doses from gamma rays (Table

5). Under the optimizing culture condition, obtained from the previous experiments (fermentation

medium composition: (g/L) sugar 200 (equal amounts of glucose + fructose); yeast extract 10; peptone

10; KH2PO4 2; (NH4)2SO4 1; pH 7.0 and incubation temperature 30oC; incubation time 24 hrs.; and

shaking speed 150 rpm. Data in Table (5) clearly indicated that gamma irradiation potentate the

productivity of the enzyme to its maximum value (46.67U/L) post exposure to 50 Gy. The maximum

sorbitol production was 64.43g/L, and the highest GFOR total activity (46.67U/L) and gluconic acid

volumetric productivity (2.71 g/L.h) were also reached at the same radiation dose.

This enhancement of enzyme production might have been due to either, an increase in the gene

copy number or the improvement in gene expression, or both (34). A gradual decrease in the enzyme

activity after exposure to the different irradiation doses at 0.25, 0.5 and 1.0 kGy was observed. This

could be explained by damage or deterioration in the vitals of the microorganism as radiation causes

rupturing in the cell membrane .This major injury to the cell allows the extra cellular fluids to enter

into the cell. Inversely, it also allows leakage out of ions and nutrients which the cell brought inside.

Membrane rupture may result in the death of a cell and decrease in the enzyme synthetic activity due

to radiation exposure (35).

In a previous study Abd El-Aziz (36) found that of 12 mutant isolated (40-60 Gy), Aspergillus

flavus G7 at 60 Gy gave maximum kojic acid production (60.31g/l) which is 115% higher kojic acid

production than the parent strain at 60 Gy.Furthermore, the kojic acid yield of the best gamma

irradiation mutant Aspergillus flavus G7 was 117 % higher than the parent strain (Aspergillus flavus

NS9) and 12.11% higher than the Aspergillus flavus wild strain.

A range of doses (20 – 160 Gy) of gamma rays at an interval of 20 Gy were applied to induce

mutation in cells of the wild lipase producing fungal strains, Aspergillus niger, Rhizopus microsporus

and Penicillium atrovenetum. Among all the mutants tested, MBL-5 obtained at 140Gy of Aspergillus

niger strain showed highest extra cellular lipase activity (13.75 ± 0.15 U mL-1) while MBL-1

Rhizopus microsporus at 20Gy showed the lowest activity, i.e., 1.06 ± 0.11 U mL-1 (37).

Table (5): Effect of low dose levels of gamma radiation on glucose –fructose oxiodoreductase

enzyme, sorbitol, and gluconic acid production

Legend as Table 2

(a): Carbon sources were added at 200 g/ L. Initial pH 7 ± 0.2; shake flasks at 150 rpm and 30 ◦C for 24hrs.

Radiation

Dosea

X

g/L

V.PX

g/L.h

S.A

U/g

E.A

U/L

G

(g/L)

V.PG

g/L.h

0.00 Gy 55.26 2.30 13.36 35.63 56.84 2.37

20.0 Gy 56.45 2.35 12.42 32.167 56.96 2.37

30.0 Gy 56.70 2.36 9.78 34.67 57.50 2.40

40.0 Gy 58.60 2.44 9.11 41.12 62.56 2.61

50.0 Gy 64.43 2.68 8.52 46.67 65.00 2.71

75.0 Gy 60.36 2.52 7.00 33.25 63.92 2.66

0.15 kGy 42.87 1.79 6.70 25.06 43.81 1.83

0.25 kGy 38.60 1.61 8.75 28.37 39.07 1.63

0.50 kGy 30.06 1.25 8.92 25.87 29.18 1.22

1.0 kGy 20.47 0.85 10.26 23.30 21.41 0.89

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

031

Chemical analysis of hydrolyzates sugars:

The use of inexpensive substrates for the fermentation process has combined benefit of utilizing

a low-grade substrate while producing a commercially valuable product. Table 6 clear, the variation of

fermentation products obtained from the concentrated wastes hydrolyzate sugars. Our studies on the

alternative substrate to the carbon source revealed that, by chemical analysis, carob, orange, and

banana peels contained a larger concentration of soluble sugars. Carob soluble sugars (49.8 g/100g

samples), and reducing sugars represented 39.96% of the total soluble sugars. Chemical analysis of

orange and banana peels indicated that the amount of reducing sugars of orange peels (77.85%) were

larger than it in banana peels (51.62%) of the total soluble sugars (29.8, and 28.11g/ 100g samples),

respectively (Table 6).

The results are in agreement with the findings of Rivas et al. (38) who found that orange peels are

rich in fermentable sugars; that is, 0.49 g/L glucose, 0.84 g/L fructose, and 3.06 g/L sucrose. The main

sugar profile of carob according to Fletcher (39) was in the following ranges (g/kg): fructose 102–115,

glucose 33.0–36.8, sucrose 299–384.

Rice straw contains a very little amount of Z. mobilis fermentable sugars (sucrose, glucose, and

fructose) and a large amounts of xylose sugar (68.0g/100 sugars). The data of Hong et al. (40) showed

that air-dried rice straw contained low amounts of reducing sugars. This is consistent with data from a

recent study by Park et al. (41), which showed that air-dried rice straw contained minimal amounts of

sucrose and free glucose and fructose. Sirkar et al. (42) showed that the sugar yield was not affected

significantly by pretreatment methods in fruit pulps were as in peels, the dilute acid pretreatment

increased the sugar release, acid pretreatment method was found to be optimal for better yield of

fermentable sugars from fruit peels.

Acid hydrolysis was used to convert total sugars to reducing sugars (glucose and fructose). In

order to enhance the efficiency of hydrolyzate fermentation, in addition to optimization of the

pretreatment and hydrolysis process for minimizing formation of the hydrolysis byproducts

(inhibitors), it is necessary to remove inhibitors (detoxify hydrolyzates) prior to fermentation.

Evaporation is a simple procedure to remove acetic acid, furfural and other volatile components in the

hydrolyzates (43).

Table (6): Chemical analysis of hydrolyzates sugars.

Effect of agricultural resource derivative hydrolysate (ARDH):

The presence of a large amount of fermented sugars stimulate the biosynthesis of fermentation

products by Z. mobilis (Table 7). Co-culture systems (mixture of fermented sugar and wastes

hydrolyzed by equal amounts) hold great promise for GFOR enzyme production from biomass, where,

Carob with Fructose, and Carob with Glucose gave 41.87U/L and 38.29U/L, respectively. The total

activity (U/L) of GFOR enzyme was 90.80% for Carob with Fructose and 83.04% for Carob with

Glucose in comparison with the glucose fructose media. Because of the little amount of preferable

carbon sources for Z. mobilis (fructose, glucose, and sucrose), which stimulate the biosynthesis

gluconic acid and sorbitol, rice straw fermentation products was the lowest one (Table 7).

Hydrolyzate Total sugars

(g/100g)

Reducing sugars

(g/100g)

(%) of

reducing

sugars

Sucrose

(g/100g)

Glucose

(g/100g)

Fructose

(g/100g)

Banana peel 28.11 14.51 51.62 13.6 4.34 10.17

Orange peel 29.8 23.2 77.85 6.6 9.6 13.6

Carob buds 49.8 19.9 39.96 29.9 6.78 13.12

Rice straw 2.65 1.9 71.69 0.75 1.4 0.50

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

031

Several mineral compounds present in sugar cane juice and molasses are known to be inhibitors

of fermentation by Z. mobilis (44). However, the physiological effects of those mineral compounds on

the growth are scarcely described in the literature.

The various pretreatment processes, in addition to generating suitable substrates for conversion

to bio-products, typically produce a range of compounds that inhibit the organisms used for

fermentation. Z. mobilis was relatively resistant to all compounds except for hydroxybenzaldehyde.

Both xylose and glucose conversion fell in Z. mobilis with increased hydrolysate concentration over

100g/L, though Z. mobilis was by far the more robust organism (45).

Hydrolyzates of various raw materials such as agro-industrial waste have been used as the

substrate. The initial total sugar concentration of selected hydrolyzates is selected between 20 and 100

g/l to minimize the effect of the inhibitor present in it (46). Marcia et al. (47) reported that, there was

48.8% reduction in Z. mobilis ethanol production from sugar cane juice when compared to synthetic

media. Kundu and Das (48) obtained a high yield of gluconic acid in media containing glucose or starch

hydrolysate as the sole carbon source. Vassilev et al. (49) used hydrol (corn starch hydrolysate) as the

fermentable sugar to produce gluconic acid by immobilized A. niger. Rao and Panda (50) used Indian

cane molasses as a source of glucose. Mukhopadhyay et al. (51) used deproteinized whey as a nutritive

medium for gluconic acid.

Table (7): Effect of agricultural hydrolyzate on glucose–fructose oxiodoreductase enzyme, sorbitol

and gluconic acid production.

Legend as Table 2

(a): Carbon sources were added at 200 g/ L. Initial pH 7 ± 0.2; shake flasks at 150 rpm and 30 ◦C for 24hrs.

CONCLUSION

The fermentation process for sorbitol and gluconic acid production can become economically

feasible by the stable enzymatic system. The best medium obtained in this study for GFOR production

was medium III, which contains glucose and fructose by equal amounts. The pre-optimization of

GFOR production by Z. mobilis has been successfully achieved through screening of significant

parameters using Plackett-Burman design. Initial carbon source, yeast extract, peptone, pH and

shaking have been identified as influence factors that significantly affecting GFOR production in this

study.

An important aspect of this study is the possibility of application of gamma radiation in a

manner to increase the enzyme production to its maximum value at a certain dose of gamma

irradiation (50 Gy) above which the enzyme activity gradually decreased. Acidic hydrolysis of wastes

a Carbon Source

g/L

X

g/L

V.PX

g/L.h

S.A

U/g

E.A

U/L

G

g/L

V.PG

g/L.h Fructose +Glucose 64.00 2.67 8.415 46.11 65.33 2.72

Carob +Fructose 56.27 2.34 8.13 41.87 55.18 2.30

Carob + Glucose 53.96 2.25 8.20 38.29 55.35 2.31

Sucrose 50.72 2.11 9.03 33.34 52.11 2.17

Carob + Sucrose 45.63 1.90 7.10 28.85 46.18 1.92

Carob 40.09 1.67 7.97 30.54 42.58 1.77

Banana Peels 38.90 1.62 4.77 24.58 37.15 1.55

Orange Peels 35.60 1.48 6.34 23.40 36.99 1.54

Rice Straw 1.55 0.06 0.26 0.95 1.49 0.06

Arab Journal of Nuclear Science and Applications, 47(4), (129-141) 2014

011

extracts is useful to convert its sugar content to reducing sugars (glucose and fructose) and convert it

to a very attractive alternative for the synthesis of high-value products from cheap and abundant raw

materials such as agriculture wastes.

From the above mentioned results, we can conclude that Z. mobilis can utilize sugars present in

agriculture wastes extract for GFOR, sorbitol, and gluconic acid production.

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