Hemicellulosic Ethanol Production in Fluidized Bed Reactor from

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Hemicellulosic ethanol production in fluidized bed reactor from sugarcane bagasse hydrolysate: interplay among carrier concentration and aeration rate Felipe A. F. Antunes, Anuj Chandel, Julio Cesar dos Santos, Thais S.S. Milessi, Guilherme F.D. Peres, and Silvio Silvério da Silva ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.7b01916 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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ACS Sustainable Chemistry & Engineering

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Hemicellulosic ethanol production in fluidized bed reactor from sugarcane bagasse hydrolysate: interplay among carrier concentration and aeration rate

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Felipe A. F. Antunesa; Anuj Chandela; Julio Cesar dos Santosa; Thais S.S. Milessia;

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Guilherme F.D. Peresa; Silvio Silvério da Silvaa.

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a

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Estrada Municipal do Campinho, s/nº - Campinho, Lorena – São Paulo, 12602-810

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Brazil.

Department of Biotechnology, Engineering School of Lorena, University of São Paulo,

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* Corresponding author: ([email protected]; [email protected]; +55

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12 31595146; +55 12 31595308)

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Abstract

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Development of efficient consolidated process is pivotal in order to design industrially

15

viable processes for conversion of lignocellulosic biomass into second generation (2G)

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ethanol. Aiming to develop process consolidation, here we explored fluidized bed

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reactor (FBR) for 2G ethanol production from sugarcane bagasse hemicellulosic

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hydrolysate (SBHH) employing calcium alginate immobilized cells of Schefferomyces

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shehatae UFMG-HM 52.2. A 22-full factorial design of experiments was carried out in

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order to evaluate the effect of aeration rate (0.027, 0.069 and 0.111 min-1) and carrier

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concentration (55.55, 83.33 and 111.11 g. L-1) on the ethanol yield (YP/S) and

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productivity (QP). Both the process variables, when used at highest level (aeration, 0.11

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min-1; immobilized carrier concentration, 111.11 g. L-1) showed maximum ethanol

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production (YP/S, 0.26 g/g and QP 0.17 g.L-1.h-1). Results showed the potential to use this

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immobilized yeast in fluidized bed reactor for ethanol production from C5 sugar

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solution. Repeated batch fermentations in FBR showed stable ethanol yield during 6

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batches (288h) followed by a gradual decrease. The use of immobilized cells in FBR

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could be conducive to the the development of viable 2G ethanol production processes.

29

To the best of our knowledge, this is the first report on 2G ethanol production from

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immobilized S. shehatae cells employing FBR using SBHH.

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Keywords: Sugarcane bagasse, Hemicellulosic hydrolysate, Fluidized bed reactor, 2G

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Ethanol, immobilized cells, Scheffersomyces shehatae

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Introduction

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Ethanol, produced from sugarcane bagasse, so called second generation (2G)

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ethanol seems a competent choice as a sustainable liquid transportation fuel particularly

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in countries like Brazil, India, China, Australia and others1. Use of 2G ethanol is not

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only important for the depletion of oil reserves, but also due to the severe environmental

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concerns such as greenhouse gases emissions and climate change

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lignocellulosic materials (e.g. sugarcane bagasse and straw, corn stover, wheat straw,

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wood and weedy materials, and others) can be converted to biofuels, their large-scale

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use in commercial ethanol production processes is rare

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China, Thailand, Pakistan, Mexico, Columbia, Indonesia, Phillipines and USA produced

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around 1600000 thousand metric tons of sugarcane annually5.

1,2

. Although

2,3.4

. Globally Brazil, India,

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In Brazil, approximately 684 million tons of sugarcane was produced in

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2016/2017 crop season 6. In general, 140kg of bagasse is generated from each ton of

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processed sugarcane7 yielding around 95 million tons of bagasse readily available for

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2G ethanol production in sucro-alcohol industries. According to Cerqueira-Leite et al. 8,

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up to 90% of the bagasse is combusted to generate electricity, which is used on site or

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distributed through the grid. Thus, the surplus of sugarcane bagasse (approximately 9.5


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million tons) could be used as a carbon source to produce (2G) ethanol and other value-

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added chemicals.

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For 2G ethanol production from sugarcane bagasse, pretreatment is necessary to

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disrupt the highly recalcitrant structure of the material to unlock complex carbohydrate

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polymers into monomeric sugars 3, 9, 10.

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Dilute acid hydrolysis of sugarcane bagasse is an efficient and fast method to de-

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polymerise the hemicellulosic fraction from cell wall into sugar monomers principally

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xylose and others sugars along with some inhibitory products which are produced due to

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sugars and lignin breakdown during the reaction 3,11.

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While the use of hemicellulosic sugars solution for ethanol production is an

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important necessity of the overall success of biorefineries, as it represents up to 30% of

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sugarcane bagasse composition 7, the microorganism for C5 sugars fermentation are

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scarce 12,13. Within this context, the search for viable microorganism that assimilates C5

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sugars remains a challenge

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Scheffersomyces shehatae UFMG 52.2, isolated from Brazilian forests has been recently

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reported as a promising ethanol producer 14,15.

13

. Among the known C5 fermenting microorganism,

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In addition to the correct selection of raw materials and microorganisms, the

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determination of processing strategies and process configurations are important for

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development of consolidated industrially feasible process 16,17.

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Hence the use of immobilized cells is advantageous due to easier recovery of the

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biocatalyst, simplified product purification, and possibility of increased biocatalyst

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concentrations, which could lead to improved process economics18.

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Different bioreactors configurations have been studied using immobilized cells

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and, amongst them, column bioreactor operated in FBR mode can be an interesting

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alternative.



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Authors have previously reported the use of S. shehatae UFMG 52.2 in free

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form19 with the goal of determining the correct fermentation medium, as well as in

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immobilized form15 with the aim of investigating the effect of cell concentration in an

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sodium alginate immobilization system. Milessi et al.20 determined the effect of stirring

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rate and immobilized cell concentration of S. stipitis (Syn Pichia stipitis). It is important

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to note that all these studies were performed in shake flasks. However, the present study

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was done at bioreactor level (2 L capacity) using fluidized bed reactor (FBR), a unique

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approach towards process consolidation for ethanol 2G production, employing

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immobilized S. shehatae UFMG-HM 52.2 to evaluate bioreactor parameters such as

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carrier concentration and aeration.

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Fluidization provide high homogeneity to the medium inside the reactor

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throughout its length, with high mass and heat transfer rates along bed, without

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mechanical impellers21. This is desirable because mechanical stirrers require higher

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energy for operation, as well as the high shear stress imposed to the reaction medium

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can disrupt cell wall of fermenting microorganisms. The proper adjustment of bioreactor

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parameters, such as aeration rate and the ratio between the biocatalyst mass and the

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reactor volume, has also presented great importance for optimizing process.

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Taking this into account, this work was aimed to investigate the use of FBR with

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a bed composed of Ca-alginate immobilized cells of S. shehatae UFMG-HM 52.2 for

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producing ethanol from sugars present in sugarcane bagasse hemicellulosic hydrolysate

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(SBHH). This is a new approach for 2G ethanol biorefineries and design of experiments

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(DOE) technique was used aiming to evaluate and understand the influence of process

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conditions in the performance of this proposed system.

99 100

Materials and Methods


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Preparation of the hemicellulosic hydrolysate of sugarcane bagasse

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Sugarcane bagasse was kindly provided by the Usina São Francisco mill,

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Sertãozinho, Sao Paulo State, Brazil. Hemicellulosic hydrolysate was obtained in 200L

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reactor, by using sulfuric acid solution (100 mg acid/g dry matter). Acid hydrolysis was

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conducted at 121°C for 20 min using a solid/liquid ratio of 1:10 for dry sugarcane

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bagasse/acid solution

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filtration. The recovered hydrolysate was then concentrated by vacuum evaporator

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under reduced pressure at 70 ∘C in a 32L capacity concentrator. The concentrated

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hydrolysate was detoxified (over liming and activated charcoal combination) according

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to the method of Alves et al. 22. The average composition of the detoxified SBHH was

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31 g.L-1 xylose, 0.3 g.L-1 glucose, and 1.6 g.L-1 of acetic acid

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medium was prepared by supplementing the sugarcane bagasse hydrolysate with 5.0

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g.L-1 (NH4)2SO4, 3 g.L-1 of yeast extract and 3 g.L-1 of malt extract 19.

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20

. The hydrolysate was separated from solid material by

20

. The fermentation

Inoculum preparation and cell immobilization

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S. shehatae UFMG-HM 52.2, yeast isolated from the Brazilian Atlantic Rain

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Forest, was kindly provided by the Microorganism Culture Collection of the Federal

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University of Minas Gerais (UFMG), Brazil. Inoculum preparation were performed

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according to the methodology of Chandel et al. 14. The culture was maintained at 5°C on

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malt extract agar slants. A loop full of a slant culture was transferred to liquid medium

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in 125-mL Erlenmeyer flasks, containing 50 mL of growth medium (30 g.L-1 xylose, 20

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g.L-1 of peptone and 10 g.L-1 of yeast extract) at 30°C, 200 rpm for 24h in incubator

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shaker (Innova 4000 Incubator Shaker, New Brunswick Scientific, Enfield, CT, USA).

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After growing, cells were centrifugated, washed and resuspended in sterile distilled

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water, for use in immobilization step.



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Cell immobilization

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S. shehatae UFMG-HM 52.2 were immobilized in calcium alginate beads, 15

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according to methodology of Antunes et al.

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suspension was added to a solution of sodium alginate (sodium salt of alginic acid from

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brown algae, Fluka analytical, Switzerland) sterilized at 121°C for 15 min, in order to

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obtain a final solution containing 1% of sodium alginate and 3g.L-1 of cells (dry

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weight). Beads of immobilized cells were produced by dripping this suspension in a 0.2

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mol.L-1 solution of calcium chloride. The beads were maintained in the CaCl2 solution

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at 4°C for 12 h. After this conditioning time, spheres having cells were washed with

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sterilized distilled water and used in fluidized bed reactor for fermentation.

. An adequate volume of the cell

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Fluidized bed reactor operation

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Column reactor of 2 L capacity (Bioengineering AG, PID Fermenter AWS,

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Wald, Switzerland), was used in all fermentation assays (540 mm × 55 mm column,

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with central vertical tube of 9 mm inner diameter). Reactor was filled with 1800 mL of

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supplemented sugarcane bagasse hemicellulosic hydrolysate added to 1 mL of antifoam

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(silicon emulsion) and recirculated in flow at 50.8 mL.min-1 (value determined by

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experimental observation of proper fluidization of beads, without need of aeration), by

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using an external pump. Different aeration rate were provided by external air pump and

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filtered before inserting in reactor, according to conditions shown in Table 1. Different

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mass of immobilized beads was added in reactor, in order to obtain carrier concentration

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(CC) of 55.55, 111.11 and 83.33 g. L-1, related to conditions of design of experiments

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described in Table 1. Figure 1 presents a schematic diagram of the proposed system.


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To investigate the influence of aeration rate (0.027 - 0.111 min-1) and carrier

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concentration (55.55 - 111.11 g. L-1) for the ethanol production from sugarcane bagasse

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in fluidized bed reactor, the main effects and their interactions of these variables were

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studied following full factorial design of experiments (22) with three replicates at center

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points. The response variables were ethanol conversion yield (YP/S) and productivity

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(QP). The analysis of statistical tests was carried out using the software STATISTICA

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for Windows (StatSoft, Inc. V.5 Tulsa, OK, USA). The process was carried out at 30 °C

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for 72h and samples were periodically collected for determination of concentrations of

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sugars and ethanol, and biomass.

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Figure 1

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Determination of minimum fluidizing velocity (Umf)

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Minimum fluidizing velocity (Umf) was determined by experimental data of

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fluid velocity and bed height, using the values of bed porosity as a function of

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superficial liquid velocity, according to the relations proposed by Richardson and Zaki

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23

.

170 171

Eq. (1) …………………………………………………….………………(U/Utc)= ε n

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where: U ... superficial velocity of the fluid; Utc ... corrected terminal velocity of the

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particle; n ... coefficient of expansion; Ɛ … bed porosity.

174 175

Eq. (2) …………………………………… Ɛ =(Vt-Vs)/Vt=1-Ms/(rs.A.H)



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where: Vt ... total volume of the reactor; Vs ... bed volume of solid particles; Ms… Mass

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of solid particles; ... rs…density of particle; A ... reactor area traversed by the fluid; H ...

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bed height

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Fermentation assays were performed using supplemented SBHH, in all

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conditions of carrier concentration, according to Table 1. The experiments were

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conducted in reactor section of 23.75 cm2, without aeration. For calculation of the bed

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porosity (ɛ), density of 1 g.mL-1 calcium alginate beads containing the immobilized

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cells was used 24.

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Determination of volumetric oxygen transfer coefficient (KLa)

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The volumetric oxygen transfer coefficient (KLa) was determined by the

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methodology of "gassing out". Using the different conditions for carrier concentration

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(Table 1). The oxygen in the culture medium in reactor was removed by purging of

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nitrogen gas. Fluidization system was started with recirculation of supplemented SBHH

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in determined flow rate (50.8 mL.min-1), and the increase in dissolved oxygen

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concentration was monitored as a function of time using an oxygen electrode (Mettler

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Toledo, 341003037/0473401, Columbus, EUA). By integrating the oxygen balance

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equation in the liquid medium, according to Equation 3, value of KLa in each condition

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was determined.

196 197

Eq. (3) …………………………………………………………………dC/dt = KLa(C*-

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C)

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where:


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C/C*corresponds to the electrode fraction of dissolved O2 concentration in relation to

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saturation concentration.

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Repeated Batch Experiments

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The repeated batch experiments were carried out in consecutive batches with

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reuse of the immobilized cells. The reactor was initially filled with stainless steel beads

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(100g) to reduce size and increase the surface area of air bubbles, then 200 g of

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immobilized biocatalyst were added into the reactor and the aeration system was set up

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to flow rate of 200 ml/min. The system was fed with detoxified SBHH (1.8l)

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supplemented with medium composition (section 2.1) and recirculated with the flow

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rate of 50.8 ml/s. In the fermentation medium, 1.5 ml of antifoam (silicone emulsion),

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and 1.193 g of the antibiotic solution (ceftriaxone sodium hemieptahydrate) was also

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added. Each repeated batch was carried out for 48h at 30ºC. In the end of each batch,

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the reactor was discharged and a fresh fermentation medium was added to the reactor by

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a peristaltic pump. The immobilized cells in the reactor bed was used as inoculum for

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the next batch. Samples were collected periodically to determine the concentrations of

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sugars and production of ethanol and biomass.

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Analytical methods

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Sugars (xylose, glucose) and ethanol concentration were analyzed by HPLC

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(Schimadzu LC-10 AD (Kyoto, Japan) with column equipped with BIO-RAD Aminex

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HPX- 87H (300 x 7.8 mm) coupled to a detector of refractive index RID-6A, with

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eluent 0.005 M sulfuric acid at a flow rate of 0.6 mL.min-1, column temperature of 45°C



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and injected volume of 20 mL. Before passing the samples in HPLC, they were filtered

226

through a Sep Pak C18 filter.

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The concentration of free cells was determined by turbidimetry using

228

spectrophotometer (Beckman DU 640 B Fullerton, CA, USA) and correlated with the

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dry weight of cells (g.L-1) through a calibration curve. For the determination of

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immobilized cell concentrations, the methodology described by Carvalho et al.

231

followed. A known mass of Ca-alginate beads was taken in the initial and after

232

completing of each fermentation assay. The beads were dried on an absorbent paper and

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dissolved in a stirred 2.0% potassium citrate solution. The suspension was centrifuged

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(2000 × g, 15 min); and the released cells from diluted calcium alginate was re-

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suspended in water to determine the turbidity, similar to the free cells.

25

was

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Ethanol yield (YP/S) (g.g-1) was defined as the ratio between ethanol production

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(g.L-1) and available sugars consumption (Xylose + glucose concentration) (g.L-1),

238

while volumetric productivity (QP) (g.L-1.h-1) was the ratio between ethanol production

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(g.L-1) and fermentation time (h).

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Results and Discussion

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The detoxified sugarcane bagasse hemicellulosic hydrolysate (composition

243

shown in material & method) was used a carbon source for the yeast S. shehatae for

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ethanol production using FBR. FBR has high stability during the process, providing

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high mass transfer rates, besides being easy to operate and to control the parameters

246

18,21

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the drag forces and the apparent weight of particles is required.

. For this bioreactor operation in a fluidized bed configuration, a balance between

248

Operation parameters are influenced by several factors such as the porosity of

249

the particles, ratio between the diameter of the support and the column, ratio between


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17,26

250

the density of the fluid and the solids as well as flow characteristics

. Tubular (or

251

cylindrical) reactors can be operated with beds in packed or fluidized configuration.

252

When operating as a packed bed reactor, if the fluid velocity is increased, the drag force

253

will also increase until it surpasses the sum of resistance forces and, then, the particles

254

lose contact with each other and begin to move, taking a fluidized configuration. The

255

velocity level correspondent to the starting of fluidization is known as the minimum

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fluidization velocity (Umf) and is a key parameter in this type of bioreactor. Umf can be

257

defined from experimental data of bed porosity (e) as a function of superficial liquid

258

velocity 23.

259

Aiming to a preliminary understanding of the characteristics of the bed of the

260

reactor and to ensure proper fluidization by using immobilized cells, initial data were

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obtained by performing experiments of bed expansion as a function of fluid velocity

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provided by the flow recirculation, without aeration. In this case, we have observed

263

proper correlation of the experimental data using Richardson and Zaki equation

264

Thus, this equation has been used taking into account its simplicity and the method

265

indicated by Smith

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velocity and porosity (ε), as well as the minimum fluidization velocity (Umf) for the

267

different carrier concentration, were determined according to Eq. 1 and 2. Thus, the

268

minimum ascendant flow that allowed fluidization in the system was obtained as 0.146,

269

0.167 and 0.166 cm. s-1 for carrier (calcium alginate + yeast cells) concentration (CC) of

270

55.55, 83.33 and 111.11 g. L-1, respectively. Actually, CC did not influence

271

significantly the Umf, presenting proportionality between increase of mass and bed

272

expansion as a function of fluid velocity. Aiming to total homogenization of the FBR to

273

obtain a stable fluidization and proper homogenization in all studied conditions, the

274

fluidization velocity (Uf) of 2.14 cm.s-1 was used in the following experiments, value at

27

23

.

for particulate fluidization. The values for particle terminal



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275

least 13 times higher than Umf, selected by considering visual observation regarding

276

complete and homogeneous fluidization in all reactor volume. This kind of observation

277

was also reported by Ram28, who determined that bubble size increased linearly with

278

distance above the distributor and excess gas velocity, eventually enabling the gross

279

circulation of solids. Within this context, important process parameters, such as aeration

280

rate and carrier concentration, were investigated.

281

The effects of these variables in the proposed system were determined by using

282

statistical design of experiments. Full 22 factorial design with triplicate at center point

283

was carried out considering ethanol yield (YP/S) and productivity (QP) as response

284

variables, as shown in Table 1.

285 286

Table 1

287 288

A range of YP/S (range of maximum and minimum yield) of 0.07 g/g was

289

obtained; the lowest value of 0.22 g/g was calculated in assay 2 (AR of 0.11 min-1 and

290

CC of 55.55 g.L-1), while the highest value, 0.29, was determined in assay 3 (AR of

291

0.027 min-1 and CC of 111.11 g.L-1). The range of volumetric productivity QP (range of

292

maximum and minimum volumetric productivity) of 0.05 g L h was calculated, where

293

the minimum value of 0.13 g L h was determined in assay 1 (AR of 0.027 min-1 and

294

55.55 g.L-1 of CC), while the maximum value of 0.18 g L h was obtained with operating

295

parameters as described in assay 4 (AR of 0.11 min-1 and 111.11 g.L-1 g of CC).

296

Carrier concentration and aeration rate are critical process variables in operation

297

of FBRs 18, 29, since small variations in supply and distribution of oxygen can influence

298

directly certain parameters in bioreactors, for example, the volumetric oxygen transfer

299

coefficient (KLa). KLa value was of 2.20, 5.44, 2.20; and 4.22 min-1, for experiments 1,


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300

2, 3 and 4, respectively, were verified. For experiments correspondent to the center

301

points, average value was 4.08 min-1. Experiments at higher aeration (Exp 2 and 4)

302

showed higher value of KLa compared to other assays, since larger aeration rate

303

provided larger contact area between the air and fluid, resulting in increased oxygen

304

transfer in the fermentation medium

305

between aeration rate and KLa, observing a reduction of approximately 10 times in KLa

306

values (from 43 h-1 to 4h-1 when aeration was 10 times reduced), in xylitol production

307

from SBHH by using Candida guilliermondii. In our work, regarding to concentration

308

of carrier in the reactor, this variable did not influence the oxygen transfer in the system,

309

considering its slight influence in KLa values.

30

. Molwitz et al.

31

also reported the relation

310

Rodmui et al 29 reported that aeration is one of the main parameters to be studied

311

in process involving C5 sugars assimilation by yeasts, since their levels may influence

312

and drive the use of substrate in the cellular metabolism. In our investigation, sugars

313

consumption was higher than 90% in all experiments. Experiments 2 and 4 showed

314

maximum ethanol concentration after 36 h of fermentation. On the other hand,

315

experiments 1,3,5,6 and 7 presented maximum ethanol concentration after 48h of

316

fermentation. Time reported to maximum ethanol concentration (Exp.1 - 4.7 g.L-1;

317

Exp.2- 6.3 g.L-1; Exp. 3 - 7.3 g.L-1; Exp. 4 - g.L-1 g.L-1 and center points - 6,97 ±0,58

318

g.L-1) was related with conditions of AR and CC. In addition to ethanol, xylitol

319

production was detected for all experimental conditions. Polyol concentrations of 7.3,

320

5.2, 1.2 and 0.7 g.L-1 were achieved in assays 1, 2, 3 and 4, respectively, while the

321

assays correspondent to the center points showed 6.9 ±0.58 g.L-1 of this polyalcohol

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production.

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concentrations of carrier. Xylitol production was affected by oxygen availability15.

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Thus, the importance of fermenter operating parameter settings was highlighted.

Highest xylitol concentrations were obtained when using lower



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The results of the design of experiments were statistically analyzed, and Figure 2

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shows the Pareto diagram, while the analysis of variance (ANOVA) is shown in Table

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2, allowing the statistical evaluation of the effects of studied dependent variables, for

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the response answers variables YP/S and QP.

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Figure 2

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Table 2

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At 95% of confidence level, only the variable aeration rate was significant (p

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Hemicellulosic Ethanol Production in Fluidized Bed Reactor from

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