Ecofriendly green conversion of potato peel wastes to high productivity bacterial cellulose

Accepted Manuscript Title: Ecofriendly green conversion of potato peel wastes to high productivity bacterial cellulose Authors: Mohamed Abdelraof, Mohamed S. Hasanin, Houssni El -Saied PII: DOI: Reference:

S0144-8617(19)30107-9 https://doi.org/10.1016/j.carbpol.2019.01.095 CARP 14553

To appear in: Received date: Revised date: Accepted date:

16 October 2018 30 December 2018 26 January 2019

Please cite this article as: Abdelraof M, Hasanin MS, El -Saied H, Ecofriendly green conversion of potato peel wastes to high productivity bacterial cellulose, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.01.095 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Ecofriendly green conversion of potato peel wastes to high

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productivity bacterial cellulose

Chemistry Department, National Research Centre, 12622, Dokki, Cairo,

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aMicrobial

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Mohamed Abdelraofa, Mohamed S. Hasaninb* and Houssni El -Saiedb

Egypt. b

Cellulose and Paper Department, National Research Centre, 12622, Dokki, Cairo,

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Egypt.

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Highlights: 

Overcome the high BC production cost and decrease the waste of resources and environmental pollution.

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Demonstrated the PPW has great possibility produce BC and mild nitric acid is an effective pre-treatment tool in biomass pre-treatment technology. Produce nitric acid PPW-hydrolysate is encouraging for thecost-effective industrial

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production of BC. Produce BC from PPW media has excellent characters.

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Abstract

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Potato peel waste (PPW) is employed as the first report on bacterial cellulose

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(BC) production by Gluconacetobacter xylinus. Scharification of PPW was performed

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by 2M different mineral acids individually. The suitable pre-treatment conditions were determined by reducing sugar release. Although all acid PPW-hydrolysates

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culture media are studied to produce BCs. Nitric acid hydrolysate gives the high productivity value The influence of nitric acid PPW-hydrolysate culture condition

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parameters were applied throughout the Taguchi method and the optimum conditions for the highest BC yield (4.7 g/L) was observed after 6 days at 35 oC, pH 9, medium

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volume 55 ml and with 8% inoculum size. The instrumental analysis of PPW-BC, included FT-IR, Particle size distrubutin, BET, DSC, XRD and SEM are cleared that the PPW-BC recorded high crystalliny82.5 %, excellent PDI. In general, this study revealed that nitric acid PPW-hydrolysate could be used as cost effective alternative

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medium for production of BC with sustainable processes that can overcome the environmental pollution. Key words: Agriculture wastes; Biotechnology; Bacterial cellulose; and Statistical analysis.

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Corresponding author:

1. Introduction

production and use of

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The development of natural biopolymers

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Mohamed S. Hasanin Cellulose & Paper Dept., National Research Centre, El-Buhouth St., Dokki, 12622, Egypt email: [email protected]

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biodegradable materials is the field of green chemistry and biotechnology which has

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been seen as a method to improve the efficiency of classical processes and encloses

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the safety of the environment. Cellulose concerned as the extremely important biodegradable polymer which produces from different sources. In addition, it is one of

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the most abundant macromolecule on earth (Klemm, Schumann, Udhardt, & Marsch,

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2001; Sheykhnazari, Tabarsa, Ashori, Shakeri, & Golalipour, 2011). Naturally cellulose found in the plants but have several drawbacks due to hazard chemicals

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which are employed with its extraction processes. Cellulose production is mainly by vascular plants, a different way that utilized to produce from another resource such as bacterial systems (Brown Jr, Millard, & Campagnola, 2003; Castro et al., 2012; W.-C.

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Lin, Lien, Yeh, Yu, & Hsu, 2013; Sheykhnazari et al., 2011). Some bacterial strains have capacity to generate the cellulose called bacterial (BC) with higher purity grade. During this process, bacteria are utilized as the nutrients to cellulose biosynthesis. Hence, the cost of the nutrients plays a crucial role in the production process economics. Some types of bacterial have capability to produce cellulose with the 3

identical to cellulose synthesized by higher plants and algae, but overall, it displays extra chemical pure cellulose. Because of its unique physical, chemical and mechanical properties that include high crystallinity, high water holding capacity, large surface area, elasticity, mechanical strength and biocompatibility, thus BC has potential applications in edible packing as food contact packaging material. In

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medicine, as wound dressing materials, artificial skin, vascular grafts, scaffolds for

tissue engineering, artificial blood vessels, medical pads, dental implants (Esa,

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Tasirin, & Rahman, 2014). In other industries products, as sponges to collect leaking oil in drug delivery agents, capsule shells, oil spill cleanup sponge mineral and oil

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recovery leather products, sports items ultra-filters for water purification audio

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speaker diaphragm plywood laminates specialty papers, polyesters automotive,

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aircraft bodies, materials for absorbing toxins and optoelectronics materials (liquid

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crystal displays) (Jozala et al., 2016). Several genera which have shown to be able to synthesize cellulose involve Sarcina, Agrobacterium, Rhizobium, and Acetobacter

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also called Gluconacetobacter. The most potent producer of BC, a Gram negative and acetic acid bacterium Gluconacetobacter xylinus could be the model microorganism

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for basic and applied studies on cellulose. G. xylinus produces an extra-cellular gellike material or pellicle, which consists of a random assembly of cellulose ribbons,

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composed of a number of micro-fibrils. This organism and its product were first identified and characterized over a century ago (J. Li et al., 2012).

A three-

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dimensional interconnected reticular pellicle is formed under static fermentation, whereas agitated and stirred conditions provide an irregularly shaped, sphere-like cellulose particle (Mohammad kazemi, Azin, & Ashori, 2015; Moosavi-Nasab & Yousefi, 2011). However, the high cost of fermentation media has limited the economic production of BC, as such media account for 30% in the total production

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cost (Rivas, Moldes, Domıń guez, & Parajó, 2004). Therefore, finding new costeffective culture media to get the maximum yield of BC in large-scale industrial applications is a critical factor. The Hestrin, & Schramm (HS) medium is usually employed in the cultivation of BC (Hestrin, & Schramm, 1956). However, this medium is costly and needs supplemental products, such as glucose, yeast, peptone,

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and etc. Therefore, wider uses of BC are determined by practical considerations with regard to scale-up capability and production costs (Cavka et al., 2013; Huang et al.,

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2014; Shi, Zhang, Phillips, & Yang, 2014). Recently, reports have focused on many different cellulose producing bacterial strains, low-cost nutrient sources, and

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supplementary materials for the biosynthesis of inexpensive BC (Kiziltas, Kiziltas,

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Bollin, & Gardner, 2015). Different waste materials from agricultural and industrial

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activities have already been studied as a way to enhance the yield and reduce the

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expense of BC production, like dry olive mill residue (Gomes et al., 2013), sugarcane molasses (Jozala et al., 2016; Tyagi & Suresh, 2016), waste beer yeast (D. Lin,

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Lopez-Sanchez, Li, & Li, 2014), wastewater from candy processing (Li et al., 2015), wood sugars (Kiziltas et al., 2015), waste from fruit processing (Hungund & Gupta,

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2010), lipid fermentation wastewater (Huang et al., 2014), rice bark , konjac powder (Goelzer, Faria-Tischer, Vitorino, Sierakowski, & Tischer, 2009), cotton-based textile

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waste (Jeihanipour, Karimi, & Taherzadeh, 2010), and coffee bean husks (Rani & Appaiah, 2013). The application of such materials could increase the sustainability of

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BC production along with reduce environmental pollution associated with the disposal of above mentioned industrial wastes. The potential of BC goes beyond existing applications, particularly if produce in considerable amounts from low-cost feedstock (Cavka et al., 2013). In this way, the high value utilization of agricultural waste is considered beneficial in terms of economics, environment and practicality (El-

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Henawy et al., 2018). Potato peel waste is a zero value waste, which occurs in large quantities after industrial potato processing and can range from 15 to 40% of initial product mass, with respect to the peeling method. Food waste utilization causes great importance in food industry and several scientific works were focused on this topic within the last years offering solutions and original approaches. In developed

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countries around 69.5% (in 2012) of total produced potatoes are processed

(Arapoglou, Varzakas, Vlyssides, & Israilides, 2010). Every year huge amount of

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PPW as being a waste remains after industrial potato processing. PPW is not ideal for non-ruminants without further treatment because it is too fibrous to become digested

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(Liitiä et al., 2003), but because a relatively inexpensive waste it includes a large

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volume of starch, non-starch polysaccharides, lignin, polyphenols, protein and small

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amount of lipids. This makes it a cheap and valuable base material for fermentation

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processes. Many studies were created and reports were concerned with PPW application possibilities in order to reduce industrial waste amount and find suitable

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application for PPW as a by-product (Arapoglou et al., 2010; De Sotillo, Hadley, & Holm, 1994a, 1994b).

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Therefore, the aim of the present study is focused on the bacterial cellulose

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production by using Potato Peel Waste hydrolysate as culture media without any extra supplementation and to investigate the effect of this different culture medium on the chemical and physical characterizes of BC network structure. Green environmental

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friendly method without any by-product waste is also purposed.

2. Materials and Methods: 2.1. Materials

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Potato peel waste (PPW) was resulting from potatoes processing, and collected from the disposal of free markets. The bright PPW without disease symptoms were selected, then washed thoroughly with distilled water. All reagents, medium and its components are purchased from modern Lab Co, India in analysis

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grad without any purification required. 2.2. Methods

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Strain and culture conditions

The BC-producing strain Gluconacetobacter xylinum ATCC 10245 is employed throughout this work; has been obtained from the American Type Culture

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Collection (ATCC), Manassas, VA, USA. The pre-culture is activated on pre-

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cultivation medium (g/L, mannitol 25, peptone 5, yeast extract 3) at 28°C and 150

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rpm for 48 h. The culture is propagated by inoculation of 3% (v/v) from mannitol

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broth to HS medium (%, w/v): glucose, 2.0; peptone, 0.5; yeast extract, 0.5; disodium

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phosphate, 0.27; citric acid, 0.115. The pH was adjusted to 6.0 at 30ºC for 24 h (Hestrin & Schramm, 1954). Then, seed culture is translated into the PPW acid

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hydrolysate and the static fermentation is carried out at 30°C for 96 h.

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Preparation of potato peel waste acid hydrolysate Acid treatment of dry PPW was performed according to method described by

Arapoglou et al, 2010 (Arapoglou et al., 2010). 40 gram of PPW containing 85%

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moisture is added in a 250 ml round-bottom flask to reach a final concentration of 30% (w/v), which are regarded as untreated PPW mixed liquor. Mixed liquor PPW (10%) is added together with 20 ml of 2.0 M of each of nitric, sulfuric, hydrochloric and phosphoric acid at100oC for 2, 3, 4 and 6 hrs. After each pre-treatment, the pH of each mixture was neutralized with 1 M NaOH to 6.0, followed by centrifuging at 7

6000g for 15 min to remove sediments and impurities. Finally, the clear supernatant is adjusted to determination of reducing sugar concentrations after sterilization at 121oC for 15 min. The total reducing sugar concentration is determined by the DNS method Miller, 1959 (Miller, 1959) with D-glucose as standard. Sugar yield is calculated as

Reducing sugar yield (%)= Reducing sugar weight (g) x100 PPW weight (g)

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Evaluation of PPW as an alternative medium for BC production

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reported by Lin et al, 2014 (D. Lin et al., 2014) using the following equation:

To evaluate the effect of PPW acid hydrolysates on the BC production by G. xylinum, the pre-inocula were prepared as described in section 2.1. The fermentation

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condition is adjusted to the same conditions of the standard medium HS: inoculum

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size 4 % (106 CFU/ml), initial pH 6.0, cultural temperature 30°C and incubation under

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static condition for 4 days. BC production, cell growth, sugar utilization and the pH of

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the remaining medium is measured along with the fermentation process. The number

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of viable cells was determined by counting colonies formed on the HS agar surface at 30°C at 48 h. as adapted by Kouda et al., 1997 andNaritomi et al., 1998(Kouda,

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Yano, & Yoshinaga, 1997; Naritomi, Kouda, Yano, & Yoshinaga, 1998).The first batch of experiments in which G. xylinus is grown on PPW hydrolysate diluted

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medium (2x, 3x and 4x) and undiluted medium compared with HS medium. All the experiments are carried out in triplicate. For each acid treatment a sample which is not

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diluted used as control. Quantification and purification of the BC At the end of cultivation period, the fermentation broth and BC were separated by centrifugation at 10,000 g for 10 min to separate cellulose from the supernatant. The supernatant is collected, and the residual sugar levels are determined as previously 8

mentioned, and the fermented media subjected to analysis of final pH. The produced BC is collected, rinses in distilled water, and immerses in NaOH 1 N at 60°C for 90 min to remove attached cells and impurities. Later, pellicles are rinsed in methanol solution and then wash with the deionized water and dry at 60°C for 24 h to evaluate the BC yield concentration in g L−1 (mass (g) of BC/volume (L) of culture medium)

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(Hong et al., 2006; Moosavi-Nasab & Yousefi, 2011). Kinetic studies

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To identify the efficiency of using substrate, the fermentation kinetics during BC

production is measured. The efficiency of BC production is evaluated after 96 h of

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cultivation and the, substrate conversion ratio, BC production rate and BC production

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BC production rate (g/L h) = mBC Vxt

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Substrate conversion ratio (%) = Si-Sf x 100 Si

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yield are calculated, respectively, as described by Lin et al, 2014(D. Lin et al., 2014):

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BC production yield (%) = mBC/ V Si-Sf

x100

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Where Si is the initial concentration of substrate (g/L), Sf is the final concentration (g/L), mBC is the amount of BC produced (g), V is the reactional volume (L) and t is

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time of reaction (h).

Evaluation effects of different parameters on cellulose production by Taguchi

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method

There are many factors affect cellulose production by Gluconacetobacter xylinus.

Design of experiment (DOE) is carried out by Taguchi method for evaluating the effects of different factors on BC production. Optimization of five factors, initial pH, medium volume (mL), inoculum size (%), Temperature (oC), and incubation

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time(day) as presented in Table 1, is studied by Taguchi method. To perform the Taguchi method, minitab17-software designed an experimental plan (L25 orthogonal array) that contains 25 experiments as shown in Table 2, where this design enable us to make more interactions between different levels of different factors to produce high yield of BC. Based on the primary results, a verification test is also applied to check

analysis of variance) for the obtained results.

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Table 1 Factors and levels which were studied by Taguchi method. Level 1 Level 2 Level 3 Level 4 Factors pH 7 8 9 10 o Temperature ( C) 25 30 35 40 Aeration (ml) 55 65 75 85 Inoculum size (%) 4 6 8 10 Incubation period 2 3 4 5 (day)

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the optimum condition. Statistical analysis is investigated by ANOVA (one-way

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Table 2 L25 array of Taguchi design of factors affecting BC production pH level Temperature Aeration Inoculum Incubation level level size level period level 1 1 1 1 1 1 2 2 2 2 1 3 3 3 3 1 4 4 4 4 1 5 5 5 5 2 1 2 3 4 2 2 3 4 5 2 3 4 5 1 2 4 5 1 2 2 5 1 2 3 3 1 3 5 2 3 2 4 1 3 3 3 5 2 4 3 3 1 3 5 3 5 2 4 1 4 1 4 2 5 4 2 5 3 1 4 3 1 4 2 4 4 2 5 3 4 5 4 1 4

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Trail No.

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Level 5 11 45 95 12 6

21 22 23 24 25

5 1 5 4 3 5 2 1 5 4 5 3 2 1 5 5 4 3 2 1 5 5 5 3 2 The numbers under factors refer to their levels; for description of levels, refer to Table 1

Characterization of BC membranes

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Fourier transform infrared (FTIR): The structure change between different produced BC is studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR)

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spectroscopy (Spectrum Two IR Spectrometer – Perkin Elmer, Inc., Shelton, USA).

All spectra are obtained by 32 scans and 4 cm−1 resolution in wave numbers ranging

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from 4000 to 450 cm−1. IR measurements which are including crystallity index (Cr.I.)

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(Nelson & O'Connor, 1964a, 1964b) and main hydrogen bond strength (MHBS)

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(Levdik, Inshakov, Misyurova, & Nikitin, 1967) are calculated.

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Differential Scanning Calorimetry (DSC) The DSC instrument (SETRAM DSC evo131, France) with scan rate 5 °C/min is used to study the thermal behavior of different

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produced BC.

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Particle size measurements The particle size distribution of the prepared samples are measured, using NicompTM 380 ZLS size analyzer, USA. Leaser light scattering is

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used at 170ᵒ.

XRD: The crystalline structure of samples is characterized using X-ray (XRD)

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diffractometer (Schimadzu 7000, Japan) operating with Cu Kα radiation (λ=0.154060 nm) generates at 30 kV and 30 mA with scanning rate of 4ºmin-1 for 2θ values between 10 and 80 degrees.

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SEM: The micrographs of the prepared samples are analyzed by scanning electron microscopy (SEM, Quanta FEG 250, FEI). To prepare the SEM sample, a thin layer of Au is coated onto the sample by sputtering coating device. 3. Results and discussion

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Evaluation of PPW acid hydrolysate as an alternative medium for BC production

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Potato peels is a zero value waste as well as the contaminant have drawback against environment which mainly consisted of sufficient quantities of starch, hemicellulose, lignin and fermentable sugars to warrant use as feedstock (Arapoglou

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et al., 2010). According to earlier reports Song et al., 2009 (Song, Li, Seo, Kim, &

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Kim, 2009), the acidic hydrolysis of starchy wastes would increase the fermentable

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sugar released for the production of microbial products (Y. Li et al., 2012). PPW acid

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hydrolysates are directly supplied to G. xylinus ATCC 10245 as carbon and nutrient

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sources to produce BC without any extra nutrient added to reduce the manufacturing costs. Different dilutions of the PPW acid hydrolysates are tested, in order to optimize

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carbon substrate concentration, and, at the same time, to reduce the effect of possible inhibitory compounds present. The results showed that the amount of favorite carbon

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source was increased by the type of acid hydrolysis and the BC production obtained using diluted PPW acid hydrolysates are higher than those from the undiluted PPW

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(Data not shown). The results indicated that the maximum BC yield is obtained in a two-fold dilution of each of nitric, sulfuric and hydrochloric acid PPW-hydrolysates, which are increased as 3.4, 2 and 1.8 folds respectively, compared to that of the PPW without dilution. This can be attributed to the fact that the undiluted samples have a high sugar concentration which could cause the inhibition of the BC production and

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reduce the supply of oxygen by the liquid medium. While in the diluted samples, the reducing sugar concentration is decreased by diluting the supernatant with water, which could lead to a better concentration for BC production (Y. Li et al., 2012; Li et al., 2015).In contrast, the diluted hydrolysate from H3PO4-treatment and the untreated PPW produced lower BC yields. Likely the cellulose production by G. xylinus in this

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case is inhibited due to the low sugar concentration present in these hydrolysate

samples, which are further diluted from the already low sugar yields of PPW obtained

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from these hydrolysates. Therefore, it was noted that, the highest sugar concentrations inhibited the BC production, which is in agreement with the findings from previous

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studies (Carreira et al., 2011; D. Lin et al., 2014). Additionally, Lin et al, 2014(D. Lin

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et al., 2014) have observed that the reducing sugar concentration has a market effect

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on BC production and would affect the fermentation cycle to some extent, due to the

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fact that the BC yields from the various sugar concentrations reach their maximum at various cultivation time. Accordingly, culture media containing the optimum diluted

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PPW acid hydrolysate resulted from each acid treatment is tested and the results of cell growth, BC yield, sugar consumption, and pH are determined at 96 h of

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bioprocess, which is considered the final time of fermentation.

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Table 3: Evaluation of BC yields with acid-PPW hydrolysate and HS culture media for 96 h: Factors

HCl

H2SO4

HNO3

H3PO4

HS

0

96

0

96

0

96

0

96

0

96

Log CFU/ml

13

15

13

14.8

13

15

13

13.5

13

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pH

6

5.5

6

5.7

6

6.4

6

5.8

6

3.8

Fermentable

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2.3

17

8.1

20

9.8

12

7.1

20

3.7

-

1.88

-

2.18

-

2.61

-

1.27

-

1.21

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Time (h)

Sugar(g/L) BC yield (g/L)

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Table 3, shows the amount of sugar and pH value in the media used to produce cellulose before and after the fermentation process compared with BC yield values. The cell growth is similar to HCl, HNO3 and H2SO4-PPW-hydrolysates, while the lowest cell growth is observed with H3PO4 and the standard medium (HS) provided moderate result. The results are described in Table 1, shows that the

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production of BC in the standard medium (HS), allow obtaining a BC production of around 1.21 g/L after 96 h of cultivation, this yield is in same range of those reported

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in the literature using the same medium (El-Saied, El-Diwany, Basta, Atwa, & ElGhwas, 2008; Kuo, Chen, Liou, & Lee, 2016). While, the BC pellicle could be

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observed on the PPW acid hydrolysate medium surface during the first two days of

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fermentation and the maximum yield of BC production is achieved using PPW-nitric

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acid hydrolysate 2.61 g/L followed by PPW-sulfuric acid hydrolysate 2.18 g/L which

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were both higher than those from the other PPW acid hydrolysate. This indicates that the BC production in PPW-nitric acid hydrolysate and PPW-sulfuric acid hydrolysate

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media is 2.2 and 1.8-fold higher than that in HS media, respectively. For a deeper understanding of the lower BC productivity in HS medium as compared with PPW

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acid hydrolysate media, we investigated the final pH and sugar consumption. As described in Table 3, at time 0, the total sugar content in each PPW acid hydrolysate

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showed different concentrations, varying from 12 to 20 g/L. The results show a similar pattern of sugar consumption for each of HNO3 and H2SO4 PPW-hydrolysate;

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however, the HCl-hydrolysate has higher sugar consumption after BC production. According to the presented data, the amount of sugars decreased from 20 g/L to 3.7g/L during the cultivation time of HS medium accompanied by sharp decrease in the final pH 3.8. Furthermore, the final pH of PPW acid hydrolysate media was close to its initial value in spite of the higher sugar consumption. Embuscado et al., 1994

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(Embuscado, Marks, & BeMiller, 1994) reported that BC production by G. xylinus will be completely inhibited at pH 3.5 and at least 80% of BC is produced at pH 6. Sugar consumption and pH drop are directly proportion with the BC productivity, since pH decrease in HS medium caused by the rapid enzymatic convert of glucose into gluconic acid by glucose dehydrogenase (GDH) of Gluconacetobacter species

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(Chen, Wang, Zhang, & Jin, 2008). It is interesting to note that, the significantly enhanced BC production in PPW acid hydrolysate medium can be attributed to the

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buffering capacity of PPW which maintains BC production in the optimal pH range

during the static cultivation period. Similarly, wastes such as corn steep liquor has

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various substances contained in them may contribute to the buffering effect may cause

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a deviation in the productivity of BC and the controllability of pH (Noro, Sugano, &

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Shoda, 2004). In general, PPW nitric acid hydrolysate medium promoted a

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considerable increase on the production of BC (2.61 g/L) when compared with other PPW acid hydrolysate results obtained in experiment as shows in Table 3.

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BC production efficiency

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Kinetic parameters are very important not just to estimate the expense of a bioprocess but additionally to develop control strategies. For this reason, the suitable

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dilutions of each hydrolysate, determined in order to used, without any supplementation of the culture media to analyze in more details the production of BC. The results obtained are compared with those obtained using the standard HS media

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Table 4.

Table 4.Comparison of Substrate conversion ratio, BC productivity rate, and BC production yield in various acid-hydrolysate and HS culture medium.

Substrate digestion with acids HCl H2SO4 HNO3

Substrate conversion ratio (%) 51.6 66.3 73.7

BC productivity rate (g/L/h) 0.0209 0.0252 0.0401 15

BC production yield (%) 32.1 22.4 32.1

H3PO4 Without (Boiling only) Control (HS)

65.09 51.4 55.5

0.0244 0.0084 0.022

25.5 24.6 14.3

Interestingly, the evolution of BC production observed for the standard HS medium along with the corresponding acid PPW-hydrolysate media considered in

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general so varied profiles. However, for nitric acid, BC production not stopped during 96 h but for hydrochloric and phosphoric acid ended in 72 h, even though the carbon

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source remains not entirely consumed. This implies that, there may be an absence of

other nutrients in the culture media in case of hydrochloric, phosphoric and untreated PPW-hydrolysate while, the continuous production of BC in case of nitric acid PPW-

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hydrolysate along with fermentation period and also the higher sugar utilization are

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closely related to the formation of sodium nitrate as nitrogen source in the culture

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medium which formed as evidence of the reaction between sodium hydroxide and

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nitric acid when adjusted the pH value at 6. In this respect, as shows in Table 4, the substrate conversion ratios, and BC production rates after 96 h for HS medium were

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55.5% and .022 g/L h respectively, however, nitric acid PPW-hydrolysate generate

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higher BC yields than HS medium with 32% and 14.3%, respectively. The acid PPW-hydrolysates (phosphoric and hydrochloric) show conversion and

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production ratios considerably lower than those observe for the nitric acid. In comparison, a considerably low BC yield is obtained for HS medium, while for nitric, hydrochloric, phosphoric and sulfuric PPW, the BC yield are (32.1%, 32.1%, 25.5%

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and 22.4% respectively) is relatively higher than for pure glucose in HS medium (14.3%). The conversion ratios (73% and 66.3%) and BC yields (32.1% and 25.5%) obtained for nitric acid and sulfuric acid PPW-hydrolysate, respectively, are higher than those of the pure HS medium. In addition, the productivity achieved in the mediumnitric acid PPW hydrolysate is approximately (0.0401 h-1) 4-fold higher than 16

that obtained in the HS medium (0.012 h−1), which contained glucose. The type of sugar present in each medium can be the main reason for the results achieve. Since PPW nitric acid culture medium shows the best results with respect to kinetic parameters, this media can be used as an alternative to produce BC. This result shows that it is possible to develop BC production by employing nitric acid PPW

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hydrolysate as the culture medium. Gomes et al., 2013 (Gomes et al., 2013) utilized dry olive mill residues for the production of BC, and the results show an increase in

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the production, with lower yields in 96-h BC yield of 0.099 g BC/gS, yield of

substrate on product.Generally, the BC production from nitric acid PPW-hydrolysate

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medium is 0401g/L h-1, which allowed these waste similar potential as carbon source

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for BC production. Moreover, the clear potential of the nitric acid PPW-hydrolysate

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for the higher production of BC compared with the pure substrates in HS medium

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resulted could be related to the rapid change in the pH value, which as pointed out above leaded to the stop of BC production. Therefore, it can be concluded that PPW

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treated with 2.0 M nitric acid at 100oC for 3h. is the most efficient treatment to improve the reducing sugar yield of PPW and supplement the culture medium with

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the nitrogen content which represented by sodium nitrate resulting from the neutralization of nitric acid with sodium hydroxide as mentioned below.

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HNO3+NaOH

H2O+NaNO3

Thus, the determination of the optimal nitric acid PPW-hydrolysate medium

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conditions with 20 g/L of the correspondent carbon source for BC production is the second step of this work. Optimization of the PPW Culture Conditions by Taguchi Design.

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For the optimal cultural conditions of PPW nitric acid hydrolysate medium, Taguchi design is applied to determine the optimum level of each of the significant parameters that brings highest BC production. The design and the response BC productivity (g/L) are shown in Table 5. The results of experiments performed in this section show that the highest average yield of BC

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was 4.72 g/L, which occurred when experiment’s conditions are as follows: The initial pH 9.0, temperature 35oC, medium volume 55 ml, inoculum size 8% and

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incubation period 6 days .This value (4.72 g/L) is approximately two-fold increase in the BC yield, when compares to (2.61 g/L), the results obtained in the nitric acid

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PPW- hydrolysate medium, without any optimization. Moreover, this result is

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approximately 4 times higher than of those observed in the HS medium (1.21 g/L).

design

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Trail No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

PT

Table 5 Effect of main parameters on cellulose production by G. xylinus using Taguchi

Final pH 6.4 6.7 6.5 6.3 6.8 8.2 7.8 7.9 7.5 8.3 8.7 8.4 8.8 8.6 8.7 10.3

Sugar consumption (g/L) 12.4 14.9 9.7 11.2 12.9 10.7 8.7 13 9.7 10.5 16.2 11.1 12.3 13.7 9.1 9.7 18

Response BC yield (g/L) 3.7 4.08 4.28 3.67 1.23 3.8 4.18 3.68 2.07 0.83 4.22 4.61 3.89 4.72 1.88 3.33

17 18 19 20 21 22 23 24 25

10.2 9.7 10.3 9.8 10.8 10.9 10.8 10.9 10.8

10.9 8.7 8.9 9.7 9.4 8.7 6.9 6.7 7.6

1.27 4.08 4.2 0.59 1.09 1.77 1.8 1.56 0.22

Table 6 Analysis of variance (ANOVA) of the calculated model for BC production

SS

df

MS

F-value

P-value Prob> F

4

4.7350

16.00

Temperature

22.5038

4

5.6260

25.79

Aeration

5.3933

4

1.3483

2.84

Inoculum size

0.7201

4

0.1800

1.73

Incubation period

1.3341

4

0.3335

0.83

Residual Error

2.3361

4

0.5840

Cor Total

51.2274

24

Model

48.87

20

34.28 34.54

0.168

16.52

0.304

6.20

0.570

8.43

A

N

0.004

9.43

M

R2 = 0.931; Adj R2 = 0.921;

0.010

%

SC R

18.9399

U

Initial pH

Contribution

IP T

Source

ED

df degrees of freedom; SS sum of squares; MS mean square;

PT

To evaluate effect of each factor on BC productivity; Taguchi design is applied to detect contribution percentage for each factor in BC productivity, Table 6, shows

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different conditions of pH and temperature has significant effects on BC productivity with contribution percentages 34.28 and 34.54 %respectively. As reported before, glucose is regarded as the most commonly sugar used for

A

the formation of BC, however this substrate generates several by-products such as gluconic acid, which results in low yields for BC. Rapid alteration in cultural pH due to production of gluconic acid is considered as a significant challenge in BC production. Therefore, controlling the pH is very difficult not just in static culture, but also in agitated culture (Bilgi, Bayir, Sendemir-Urkmez, & Hames, 2016; Shoda & 19

Sugano, 2005).Alternatively, Li et al., 2012(Y. Li et al., 2012)studied the addition of some chemicals such as ethanol and sodium citrate to the medium, which reduce byproducts. Thus, the utilization of nitric acid-PPW-hydrolysate, which has starch as the main carbon source, can both decrease the formation of byproducts and increase the yield of BC. Panesar et al. 2012 (Panesar, Chavan, Chopra, & Kennedy, 2012) have

IP T

noticed that lower pH and incubation time lead to minimum cellulose production however, cellulose production increases with an increase in incubation time and pH.

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Our results revealed that alkaline pH (8-9) was the limiting factor to BC production. This result is consistent with the work of (Pourramezan, Roayaei, & Qezelbash,

U

2009), who founded that alkaline pH is favorable for cellulose production as a result

N

of minimum conversion of glucose into gluconic acid, which increases cellulose

A

production. The availability of phenolic compounds in the PPW may be contribute to

M

its high buffering capacity and also has a good impact on the formation of biopolymer.

ED

This study demonstrated an effective way to produce BC by using culture media at an inexpensive, which can be improved by optimizing the fermentation

PT

conditions (Jozala et al., 2015). BC production from molasses and corn steep liquor to reduce the cost of culture media and achieved the maximum BC yield 2.21g/L is

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employed by Jung et al., 2010(Jung et al., 2010) while, in our study, the maximum BC yield is 4.72 g/L from nitric acid PPW- hydrolysate medium, which has been 2.2

A

times higher. Bilgi et al, 2016 (Bilgi et al., 2016) studied cost-effective culture medium using carob and haricot bean as carbon and nitrogen sources, the results indicated that maximum BC production was obtained after 7 days of cultivation period1.36 g/L. This value is approximately 3 times lower than the yield of nitric acid PPW hydrolysate culture medium. Carreira et al, 2011(Carreira et al., 2011) have

20

improved BC yield with adding yeast extract, KH2PO4 and (NH4)2SO4 into crude glycerol as supplement nutrient. The maximum BC production improvement (200%) occur with 4.8 g/L of BC yield, in the present study no extra nutrient is added. Moreover, BC production using PPW pretreated by mild nitric acid displayed a high yield and PPW hydrolysates could totally substitute the conventionally used chemical

IP T

media.

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Produced BC Crystalinity and structure

The produced BC from nitric acid-PPW hydrolysateis characterized and

U

evaluate with different instruments techniques in comparison with other which

N

produce by media HS. The obtained data have been evaluated the produced BC

M

A

characteristics and features as well as its behavior.

ED

PT

0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 4000

A

CC E

Abs.

A

HS PPW

3500

3000

2500

2000

1500

Wavelength (cm-1)

21

1000

500

B

120

Intensity

100 80

HS

60 40 20 0 0

10

20

30

40

50

60

Pos. [°2Th.]

IP T

100

PPW

60 40 20

SC R

Intensity

80

0 0

10

20

30

40

50

U

Pos. [°2Th.]

60

N

Figure (1): A) FT-IR of bacterial cellulose produced form HS media and PPW

A

hydrolysate and B) XRD pattern of bacterial cellulose produced form HS media and

ED

M

PPW-hydrolysate.

FTIR considered as an important characteristic tool for macromolecule as

PT

cellulose. Cellulose has many characteristic peaks refer to its function group (Zhao et al., 2018). In the wave numbers of 4000–400 cm-1 of the FT-IR spectrathe BC

CC E

samples are shown in Figure 1A, the characteristic bands of cellulose (type I) synthesized in the two different media nearlyappeared at the same wavenumbers of

A

the FT-IR spectra. Hydroxyl group stretching vibration, including 3429and 3439cm-1 for PPW and HS media respectively the shift of OH group to lower frequency for PPW indicated that the BC is more condense than HS and may be more crystalline this is in agreement with IR calculations which indicated that MHBS for PPW is 5.4 and for HS 5.9. Additionally for the hydroxyl groups stretching vibration of

22

methylene (–CH2–) band appear at 2935 cm-1 for both media. Also for the symmetric stretching vibration of methyl (–CH3–) recorded band for both media at 2840cm-1. However the asymmetric deformation vibration of methyl and methylene is shifted from 1428cm-1 in HS to 1400 cm-1 in PPW this emphasize that the shifting in OH stretching vibration and confirm the increase of crystallinaty in PPW than HS

IP T

bacterial cellulose (Park, Jung, & Park, 2003). The functional groups of BC products

which recorded in 1039 and 900 for the asymmetric deformation vibration of methyl

SC R

and methylene and for the stretching vibration of C–O–C in the sugar ring, respectively indicated that the formula of the produced BC is near in chemical

the culture medium shows little influence on the

N

FTIR data reported that

U

structure of nature pure cellulose(Cakar, Özer, Aytekin, & Şahin, 2014). Hence the

A

functionalgroups of BC products and PPW shows an increase in crystallinaty (Oh et

M

al., 2005). This is in agreement with the IR calculations which figure out the PPW BC

ED

is more crystalline than HS BC with Cr.I. 1.3 and 1.1 for PPW and HS, respectively.

The XRD patterns obtained from the BC samples are showen in Figure1B.

PT

The characteristic peaks that appeared at 14.5o and 22.9o stood for the crystal plane

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(1–10), and (200), respectively (Son et al., 2003). They were totally observed in all the BC samples tested, illustrating that the cellulose exhibited primarily the I-b pattern, regardless of the type of culture medium employed. However, the peak

A

intensities of BC obtained in fermentation PPW media at 14.5oand 22.9o are more than those in HS medium, indicating that the BC synthesized using PPW as culture medium has much more crystal component and less amorphous component. Crystallinity was calculated based on the Segal method (Guo, Cavka, Jönsson, & Hong, 2013) by using the peak intensities of the (200) peak (I002) and the height of 23

the minimum (Iam) between the (200) and the (110) peaks and observed that PPW media was 82.5 % and HS media 77.5%, this is value is acceptable according to other studies (Zhao et al., 2018). In our study, the above data which obtained from FTIR and XRD for BC samples cleared that the PPW media produced BC typical in

from ship, available, environmental friendly and economic source.

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Thermal behavior of produced cellulose

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structure with other which produced from HS media. However with high crystallinity,

Thermal stability and behavior of produced BC were studied with DSC and

A

CC E

PT

ED

M

A

N

U

the obtained data are illustrated in Figure 2.

24

40

30

332

HS PPW 300

IP T

10

0

124

-10

135 -20 0

100

200

300

400

∆T (°C)

Heat capacity (J/g)

123

56

117

322

312

21

-47

168

135

52

242

Stage 1

92

148

Stage 2

301

Stage 1

106

Stage 2

280

360

327

80

92

416

449

445

33

2.0

PT

Stage3

ED

PPW

M

HS

TP (°C)

N

Tf(°C)

A

To (°C)

Sample

500

U

Sample Temperature(°C)

SC R

HeatFlow(mW)

20

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Figure (2): DSC of bacterial cellulose produced form HS media and PPW hydrolysate

A

and DSC data for BC produced from HS and PPW.

DSC degradation stages showed the highly thermal stability for BC which

produced from PPW than other BC which produced from HS media as tabled in Figure 2. PPW-BC was recorded three stages as thermal response and HS-BC showed two stages only. Additionally the PPW-BC appears high activation energy 25

with exothermal degradation behavior. However HS recorded low activation energy in the first stage and endothermic degradation behavior in the second stage. The DSC data emphasized that the PPW BC showed high thermal stability in comparing with the other which produced from HS media (Barud et al., 2008).

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Surface study The isotherm charts types are used as a useful key to predict the surface and

SC R

porosity of the studied materials (El-Saied, Mostafa, Hasanin, Mwafy, & Mohammed,

2018). The isotherm chart in Figure 3 are presented both produced BC. Additionally the surface area of PPW which tabled in Figure 3 showed slightly increase than the

U

HS BC. However the pore volume of HS-BC records the higher value than PPW-BC.

N

This cleared that the amorphous region which characterized with high porosity is lead

A

to increase the pore volume in HS BC, however in case of PPW BC the crystal

M

behavior lead to increase in surface area and decrease in pore volume.

ED

A

A

CC E

PT

B

Sample

VT(0.95),

SBET , m2/g

Smes(BjH),cm3/g

Vmeso, cm3/g

Pore Redus, nm

cm3/g

HS

6.25

6.39*10-3

3.37

0.0054

2.05

BC

6.79

6.18*10-3

3.10

0.0048

1.82

Figure (3): Complete isotherm for produced bacterial cellulose, A, B are from HSmedia and BC from PPW-hydrolysate media and surface area and BET calculations of produced BC.

26

Particle size distribution The Polydispersity index PDI is a measure of dispersion homogeneity (Hasanin, Mostafa, Mwafy, & Darwesh, 2018). PDI value is ranged from 0 to 1.0 where from 0 to 0.3 is refer to the highest homogeneity distribution state (Cheng, Zhao, & Wu,

IP T

2018). The narrow uniform Gaussian distribution can be pointed to high PDI for HS, and PPW where is the PDI value of HS and PPW- BCs recorded 0.145 and 0.195, respectively. This is referred to highly homogeneity in particle of produced BCs as

SC R

well as emphasized the uniform 3-D network structure. The mean diameters were

measured in aqueous solutions with relative concentration as 1003.4 and 696 nm for

U

HS, and PPW particles,respectively. Crystalline properties of PPW-BC which are

N

discussed in crystallinity part were emphasized the particle size measurements.

A

Morphological microstructure of the BC produced

M

The morphological studied of produced BC was carried out on the two different

ED

culture media with using HR-SEM to focus closely on the fibers via 6000x magnification and illustrated in Figure 4. HS-BC fibers observed as flat fibers

PT

accumulated with amorphous blocks which are reduced in PPW-BC case. The fibers appear more width and flat as well as more topography homogenous in PPW-BC.

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This refers to organized fibril network(Adall, 2016). This results are emphasized the high crystallinity of PPW-BC and particle homogeneity as well as the high surface

A

area.

27

IP T

C

A

A

N

U

SC R

D

B

4. Conclusion

ED

from PPW-hydrolysate media.

M

Figure (4): SEM for produced bacterial cellulose, A, B are from HS-media and BC

Our findings could not only overcome the high BC production cost, but also

PT

decrease the waste resources and environmental pollution. Therefore, it has

CC E

beennotonlydemonstrated that PPW has great possibility produce BC, but also that mild nitric acid is an effective pre-treatment tool in biomass pre-treatment technology. Additionally, the resultsobserved herein for nitric acid PPW-hydrolysate is

A

encouraging for the cost-effective industrial production of BC. Finally, the instrumental analysis emphasized that the produced bacterial cellulose from PPW media has excellent characters which included highly surface area, homogenous particle according PDI value, high crystal structure and homogenous network as illustrated from SEM topography. 28

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