Acetone-butanol-ethanol (ABE) production from fermentation of hot

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Acetone-butanol-ethanol (ABE) production from fermentation of hot-water extracted hemicellulose hydrolysate of pulping woods Wenjian Guan, Guomin Xu, Jingran Duan, and Suan Shi Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03953 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Industrial & Engineering Chemistry Research

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Acetone-butanol-ethanol (ABE) production from fermentation of hot-water

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extracted hemicellulose hydrolysate of pulping woods

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Wenjian Guan1, Guomin Xu1, Jingran Duan1 and Suan Shi2*

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1. Department of Chemical Engineering, Auburn University, Auburn, AL 36849, United States

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2. Department of Biosystems Engineering, Auburn University, AL 36849, United States

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* Corresponding author. Email address: [email protected]

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ACS Paragon Plus Environment

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Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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In Kraft pulping process, hemicellulose portion of wood is usually discharged as a waste

27

stream into the black liquor, representing a highly underutilized sugar source. In this

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study, the hemicellulose prehydrolysate is investigated as a liquid sugar source for

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production of acetone, butanol, and ethanol by ABE fermentation. For this purpose, hot-

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water treatment is applied to Poplar (hardwood) and Southern pine (softwood) for

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hemicellulose extraction. The acquired hemicellulose prehydrolysate was analyzed to

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contain, in addition to the carbohydrates in the form of oligosaccharides, various

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degradation compounds. The toxicity test with model compounds indicates phenolic

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compounds exert tremendous inhibition on the cell growth. Therefore, detoxification is

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required prior to fermentation. Adsorption with activated carbon is an effective

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detoxification method greatly reducing the phenolic content and alleviating the phenols-

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induced inhibition. Upon detoxification, simultaneous saccharification and fermentation

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(SSF) of concentrated poplar prehydrolysate with 43.3 g/L of sugar produced a total of

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10.8 g/L ABE giving a solvent yield of 0.25 (g-solvent/g-sugar). Comparatively, SSF of

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concentrated southern pine prehydrolysate with 46.6 g/L of sugar resulted in an ABE

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production of 13.2 g/L and productivity of 0.28 (g-solvent/g-sugar). The details of hot-

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water extraction conditions, the performance of detoxification as well as the fermentation

43

profiles are discussed. The technical feasibility of utilizing the hemicellulose

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prehydrolysate as feedstock for biobutanol production has proposed an example of the

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concept of an integrated biorefinery.

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Keywords: Acetone-butanol-ethanol (ABE), hydrolysate, Clostridium acetobutylicum

hot-water


treatment,

hemicellulose

2

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1. Introduction

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Pulp and paper mills project a great opportunity for integrated forest biorefinery

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to produce, in addition to pulp and fiber products, value-added biochemicals with

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established infrastructure and technology

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woodchips are fractionated into a major product of pulp, generating a number of waste

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products ending up as a complex mixture in the black liquor, including extractive,

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hemicellulose-degraded carbohydrates, and lignin derivatives 2-5. For the need of pulping

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chemical (NaOH and Na2S) regeneration, black liquor is concentrated, and hemicellulose

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and lignin fraction is incinerated in the recovery boiler for energy and power generation 2.

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The facts that large quantities of carbohydrate waste materials (4-5 million tons per year)

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are produced from the pulp and paper industry in the US and the heating value of

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hemicellulose (13.6 MJ/kg) is relatively low in comparison with that of lignin (27.0

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MJ/kg) makes the hemicellulose portion of the pulping wood are adequately available 2-4,

61

6

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therefore been proposed to diversify the product portfolio and generate extra revenue for

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the pulp mills.

1, 2

. In a traditional Kraft pulping process,

. The idea of utilizing this sugar source for value-added biochemical production has

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Biobutanol is one of such high-value products with versatile industrial

65

applications and vast global market demand. The market for butanol is rapidly grown at a

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rate of 120,000 MT per year, forecasting a market of USD 9.9 billion by 2020

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Fermentative production of butanol has typically been performed with solvent-producing

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Clostridium species with acetone, butanol, and ethanol (ABE) as major fermentation

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products. One of the attractive features about ABE fermentation is that the wild-type

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Clostridium culture is capable of catabolizing both hexoses (e.g., mannose, the leading


7, 8

.

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component in softwood hemicellulose) and pentose (e.g., xylose, the dominant

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component in hardwood hemicellulose) for solvent production

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papermaking industry generally combines hardwood and softwood pulp together for a

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desired paper property (e.g., strength, brightness, or so) makes both types of

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hemicellulose abundantly available yet underutilized in pulp mills.

9, 10

. The fact that

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Given the complex nature of black liquor, it is rather challenging to recover the

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hemicellulose from black liquor. Kudahettige-Nilsson et al. investigated hemicellulose

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recovery from birch Kraft black liquor via acid precipitation and found that significant

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portion of solubilized polysaccharides was degraded into hydroxyl carboxylic acids under

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the alkaline condition

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ABE production resulted in a far lower yield (0.12 g-solvent/g-sugar) than that of xylose

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control (0.34 g-solvent/g-sugar). The presence of toxic phenolic compounds from lignin

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degradation and carboxylic acids from polysaccharide degradation collectively led to the

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inferior fermentation 2, 12.

11

. In their study, fermentation of the recovered hemicellulose for

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Implementation of a pre-extraction (also named as pre-hydrolysis) step prior to

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the chemical pulping process for hemicellulose recovery has been a more common and

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practical approach

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specifically developed for the production of dissolving pulp with more than 90%

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cellulose by applying hot-water or dilute acid or alkali solution 4, 5, 13. A key criterion for

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hemicellulose extraction is not to cause measurable degradation on the subsequent pulp

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quality and yield. As a more frequent practice, hot-water pre-extraction typically imposes

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slight damage on the subsequent pulp yield and property in comparison with dilute acid

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treatment while at the same time, generating concentrated hemicellulose prehydrolysate

2, 4

. Existing technologies for hemicellulose extraction have been


4

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with fewer amounts of lignin-degraded and inorganic microbial inhibitors than dilute

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alkaline extraction 2, 4, 13.

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Helmerius et al. reported the use of hot-water extracted prehydrolysate of silver

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birch (hardwood) as a sugar source for succinic acid production with the genetically

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modified culture of Escherichia coli AFP 184 2. Similarly, Kang et al. reported

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bioconversion of the hemicellulose prehydrolysate of southern pine (softwood) into 14

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bioethanol with the yeast culture of Saccharomyces cerevisiae ATCC 200062

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Detoxification of the hemicellulose prehydrolysate with either overliming or charcoal

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adsorption, however, was commonly required in their study

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water treatment generates various types of toxic compounds for the microbial

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fermentation (e.g., aliphatic acids, sugar-degraded furan derivatives, and lignin-degraded

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phenolic compounds) 9, 15-17. Sun and Liu previously investigated the utilization of dilute

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acid-hydrolyzed hemicellulose hydrolysate of sugar maple as feedstock for ABE

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production

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combination of overliming and nano-filtration was applied to ensure a solvent production

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of 11 g/L of ABE, without which only 0.8 g/L of ABE could be produced 15.

15

2, 14

.

. It is known that hot-

. In their study, detoxification of hemicellulose hydrolysate with a

110

The present study seeks to investigate the use of hemicellulose prehydrolysate of

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two types of pulping wood: hybrid poplar (hardwood) and softwood pine (softwood) as

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feedstock for biobutanol production. The chemical compositions of the prehydrolysate

113

(i.e., carbohydrates and degradation products) from hybrid poplar and southern pine were

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comparatively presented, followed by a toxicity test of the model phenolic compounds.

115

The effectiveness of two types of detoxification treatment (overliming and adsorption)

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was discussed in conjunction with the fermentation results.


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2. Materials and methods

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2.1 Pulping woods, enzyme, and microorganism

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Debarked hybrid poplar was collected from a forest products laboratory at Auburn

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University (Auburn, AL) and southern pine was provided by Rock-Tenn Co (Demopolis,

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AL). Two types of raw woodchips were grounded and screened for an average particle

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size of 1×1×0.5 cm. The woodchips were then stored at 4 ºC. Cellulase (C-Tec 2, Lot No.

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VCNI0001) with a protein content of 255 mg-protein/mL was supplied form Novozymes,

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North America (Franklinton, NC). Multifact xylanase with a protein content of 42 mg/mL

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and Multifact pectinase with a protein content of 82 mg/mL was gifted from Genencor

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(Paulo Alto, CA).

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2.2 Hemicellulose extraction with hot-water treatment

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Hemicellulose extraction was performed with hot-water treatment in a 4 L Parr reactor

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(Parr Instrument Co., Moline, IL) under the following conditions: the liquid-to-solid ratio

130

of 5, 170 ºC, and 1 h. The conditions of hot-water treatment were referenced from a

131

previous study, under which the resulting pulp quality and yield were not subjected to

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sizable degradation 14. The Parr reactor was equipped with a temperature controller and a

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motor-driven stirring bar. Prior to the hot-water treatment, 500 g (oven-dried weight) of

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woodchip was pre-soaked overnight at room temperature in 2.5 L DI water. Upon the

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completion of hot-water treatment, the liquid portion was separated from residual solids

136

via vacuum filtration and collected as prehydrolysate. The dilute prehydrolysate was then

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concentrated in 1 L round-bottomed flask with Rotavapor (Büchi RE-121, Switzerland)

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under the following conditions: rotating speed of 80 rpm, 420 mm Hg vacuum and 65 °C


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(water bath). The chemical compositions of the prehydrolysate and the hot-water treated

140

solids were both analyzed.

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2.3 Detoxification of concentrated hemicellulose pre-hydrolysate

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Overliming treatment or charcoal adsorption was applied to improve the

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fermentability of hemicellulose prehydrolysate. The pH of concentrated poplar pre-

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hydrolysate/southern pine prehydrolysate was 3.3/3.4. Overliming was performed with

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the addition of calcium oxide (CaO) to adjust the pH of the prehydrolysate near 10. The

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mixture was then incubated at 60 °C, 150 rpm. Upon 6-h incubation, the pH of

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prehydrolysate decreased to near 6.7. The lime-treated prehydrolysate was then

148

centrifuged for precipitates removal.

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Charcoal adsorption treatment was performed in batch-mode with a charcoal

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particle size of 20-40 mesh. Before the treatment, charcoal was rinsed with DI water on a

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filter paper to remove the impurities and oven-dried overnight at 45 °C. The adsorbents

152

were loaded in the prehydrolysate at 5% (w/v). The mixture was then incubated at 60 °C

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and 150 rpm for 6h. The detoxified prehydrolysate was recovered via centrifugation.

154

Following adsorption treatment, the pH of the detoxified prehydrolysate was adjusted to

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6.5-7 with the addition of calcium carbonate (CaCO3). The chemical composition of

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detoxified prehydrolysate was analyzed according to NREL/TP-510-42623.

157

2.4 Microorganism and culture preparation

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The microorganism (Clostridium acetobutylicum ATCC 824) for the ABE fermentation

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was purchased from American Type Culture Collection. The spore suspension of C.

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acetobutylicum was maintained at -20 ºC on Elliker Broth (BD DifcoTM) with the addition


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of glycerol (20 wt.%). The method for culture maintenance and inoculum preparation has

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been detailed in previous study 10.

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2.5 ABE fermentation with pure sugar as feed and toxicity test

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The fermentation tests in the present study were carried out anaerobically at 36 °C

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at 150 rpm in 125 mL serum bottle with a working volume of 50 mL. The P2 medium

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was chosen as the fermentation medium. The detailed procedure for ABE fermentation

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with pure sugar as the substrate has been described in previous study 10. The toxicity test

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was performed with the addition of various model phenolic compounds in the

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fermentation with pure sugar as feed. A total of four model compounds were tested,

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including p-coumaric, ferulic acid, vanillic acid and 4-hydroxybenzoic acid. Briefly, the

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individual inhibitor was added in the fermentation broth at the level of 0.25-2 g/L. The

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cell growth (OD600) was measured in the stationary phase after 36-h anaerobic

173

incubation.

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The formula of P2 medium is as followings (g/L): KH2PO4, 0.5; K2HPO4, 0.5;

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MgSO4·7H2O, 0.2; FeSO4·7H2O, 0.01; MnSO4·H2O, 0.01; NaCl, 0.01; ammonium

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acetate, 2.2; yeast extract, 1.0.

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2.6 Simultaneous saccharification and fermentation of hemicellulose pre-hydrolysate

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Simultaneous saccharification and fermentation (SSF) was used as the

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bioconversion strategy for biobutanol production from hemicellulose prehydrolysate. The

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prehydrolysate was filter-sterilized by passing through a 0.45-µm syringe filter (VWR)

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and then collected into a sterilized serum bottle. Calcium carbonate (5 g/L) was added as

182

an extra buffer to maintain the pH of the broth during fermentation. The organic and


8

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mineral nutrients of P2 medium were directly supplemented to the prehydrolysate.

184

The enzyme was then added as follows. For the xylan-rich poplar prehydrolysate,

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a combination of cellulase (5 mg-protein/g-xylan) and xylanase (25 mg-protein/g-xylan)

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was loaded, whereas an enzyme cocktail of cellulase (5 mg-protein/g-xylan), xylanase

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(10 mg-protein/g-xylan) and pectinase (15 mg-protein/g-mannan) was applied when it

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came to the prehydrolysate of southern pine with mannan as the leading component. The

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headspace of the serum bottle was flushed with nitrogen gas to develop the anaerobic

190

condition. The seed-culture was then aseptically inoculated at 6%(v/v). The bottle was

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kept sealed to maintain the anaerobic condition (N2). Aliquots of samples were collected

192

with 1.0 mL syringe at 12-h interval.

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2.7 Analytical methods

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The solid composition of woodchips before and after pretreatment was analyzed

195

according to NREL analytical procedure (NREL/TP-510-42618). The chemical

196

composition of hemicellulose prehydrolysate (hot-water extracted, concentrated and

197

detoxified) was also analyzed via secondary hydrolysis as described in the protocol

198

(NREL/TP-510-42623). Hemicellulose prehydrolysate was characterized as the

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carbohydrate content (oligosaccharide and monomeric sugar) and degradation products

200

(acetic acid, furfural, HMF and soluble lignin content). The oligosaccharide in the pre-

201

hydrolysate was calculated by subtracting the monomeric sugar content in the pre-

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hydrolysate from the total monomeric sugar content after secondary hydrolysis. The

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soluble lignin content in the hydrolysate was measured by a UV-vis spectrophotometer

204

(BioTek Instruments, VT) and reported as the optical density (OD) at a wavelength of

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290 nm.


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The sugar analysis was quantified by high-performance liquid chromatography

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(HPLC) equipped with Aminex HPX-87P column (Bio-Rad Laboratories, Hercules, CA)

208

and Refractive Index detector (Shodex, Japan). The mobile phase for Aminex HPX-87P

209

column is HPLC grade water. Other products (i.e., acetone, butanol, ethanol, acetic acid,

210

butyric acid, furfural, and HMF) in this study were analyzed by HPLC equipped with

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Aminex HPX-87H anion exchange column (Bio-Rad Laboratories, Hercules, CA) and

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Refractive Index detector (Shodex, Japan). The mobile phase for Aminex HPX-87H

213

column is 0.02% sulfuric acid. The solvent yield was calculated on the weight basis as

214

the amount of ABE production divided by the total sugar in the pre-hydrolysate.

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3 Results and Discussion

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3.1 Hemicellulose extraction with hot-water treatment

217

Composition analysis of the woodchips before and after hot-water treatment gave

218

the information about the degree of hemicellulose removal and overall weight loss of

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woodchips (Table 1). Major components of untreated hybrid poplar consisted of 42.2% of

220

glucan, 18.7% of xylan and 27.5% of lignin. The hot-water treatment resulted in a total of

221

15.7% of weight loss, primarily coming from the xylan (8% loss) and extraneous

222

extractives (acetyl, protein, etc.). The treated poplar sample, however, exhibited no

223

significant decrease in other components, particularly the glucan content (1% loss), in

224

comparison with the untreated sample.

225

Untreated southern pine was analyzed to contain 40.6% of glucan, 6.8% of xylan,

226

9.6% of mannan and 32.8% of lignin. The components of the hemicellulose portion of

227

southern pine are dramatically different from poplar. The backbone of hardwood


10

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hemicellulose comprises dominantly acetylated 4-O-methyl glucuronoxylan whereas the

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leading building-block of softwood hemicellulose is glucomannan, followed by

230

glucuronoxylan

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following the hot-water treatment, which, together with water-soluble extractives,

232

collectively led to 13.6% of overall weight loss. The fact that hot-water treatment had

233

caused about 1% of loss in cellulose content indicated pre-extraction was highly selective

234

for hemicellulose portion for both the hardwood and softwood. Huang and Ragauskas

235

previously demonstrated the hemicellulose pre-extraction did not typically affect the

236

degree of polymerization (DP) of the cellulose content 4. The resulting pulp quality and

237

yield could be experimentally determined, which were not included in the present study.

238

In facts, various studies have validated the potential impacts of hot-water extraction on

239

the resulting pulp property and yield could be largely offset by optimization of a pre-

240

extraction process, or adjusting the downstream pulping conditions, e.g., cooking time,

241

pulping chemical dosage 3, 4, 18.

2, 4

. In this case, 4.3% of mannan and 2.3% of xylan were removed

242

The chemical composition of the hemicellulose prehydrolysate is presented in

243

Table 2. Hot-water treatment of hybrid poplar released a total of 22.2 g/L of

244

carbohydrates with xylose (16.8 g/L) as the dominant component in the prehydrolysate.

245

Particularly notable is that the majority of the carbohydrates were present in the form of

246

oligosaccharides (17.9 g/L) and only a small portion was monomeric sugar (4.3 g/L).

247

When it came to the southern pine, a total of 19.3 g/L of carbohydrates with 15.0 g/L of

248

oligosaccharides was acquired after hot-water treatment. In this case, mannose (8.7 g/L)

249

became the leading component, followed by xylose (4.1 g/L) and galactose (2.8 g/L). The

250

hot-water treatment works by cleaving off the acetyl groups in hemicellulose backbone,


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simultaneously releasing the polysaccharides and acetic acid in the aqueous solution 2, 13.

252

Consequently, the increased acidity further hydrolyzes the polysaccharides into oligomers

253

and monomers. The ratio of oligomer to monomer was dependent on the acidity of the

254

aqueous phase

255

generate monomer-dominant prehydrolysate but result in a great reduction in the pulping

256

yield 1, 2, 15, 16, 19.

2, 19

. Dilute sulfuric acid-assisted hot-water extraction was known to

257

The hemicellulose prehydrolysate was then concentrated to increase the

258

carbohydrates content. After concentration, the total carbohydrate in the concentrated

259

poplar/southern pine prehydrolysate was increased to 49.1/52.4 g/L (Table 3).

260

3.2 Detoxification of concentrated hemicellulose prehydrolysate

261

Albeit hot-water treatment generated the prehydrolysate rich in carbohydrate

262

content, the formation of various degradation products, including acetic acid, furfural,

263

hydroxymethylfurfural (HMF) and phenolic compounds, acted as potential microbial

264

inhibitors for the subsequent fermentation (Table 2). Studies have validated that furfural

265

or HMF from sugar degradation was not toxic to the solvent-producing C. acetobutylicum

266

in ABE fermentation

267

to have a stimulatory effect on the growth and ABE production 9, 20.

9, 15

. Indeed, the presence of furfural and HMF have been reported

268

The concentration of acetic acid in poplar prehydrolysate was 2.0 g/L, which was,

269

however, increased to 4.8 g/L after secondary hydrolysis. This indicated the dissolved

270

oligosaccharides carried a considerable portion of an acetyl group, which would be

271

released into acetic acid once the oligomer was hydrolyzed. The release of acetic acid in

272

SSF disrupts the pH of the fermentation medium, severely reducing the solvent


12

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21

273

production in ABE fermentation

274

prehydrolysate (4.8 g/L) was substantially higher than that in the southern pine

275

prehydrolysate (1.6 g/L). This was in agreement with the common notion that the

276

majority of acetyl group linked to the glucuronoxylan backbone of hemicellulose, the

277

content of which was much higher in the hardwood species 1, 22.

278

. The overall acetic acid content in the poplar

Additionally, during the hot-water treatment, lignin was partially degraded into 14, 15

279

phenolic compounds in the prehydrolysate

. Toxicity tests revealed model phenolic

280

compounds of p-coumaric, ferulic acid, vanillic acid and 4-hydroxybenzoic acid were

281

toxic to the solvent-producing Clostridia (Fig. 1). These compounds act as potent

282

inhibitors for the culture growth when their concentrations reached to 0.25-0.5g/L. The

283

culture could not survive when the concentration of ferulic acid and p-coumaric acid

284

reach to 1.5 g/L whereas a 50% reduction in the cell growth was observed with the

285

presence of 1 g/L of vanillic acid and 4-hydroxybenzoic acid. It should be noted that all

286

of the four compounds are sparingly soluble in water. The inhibition has been proposed

287

to be the hydrophobic interaction of these phenolic compounds to the cell, damaging the

288

function of cell membrane (hydrophobicity, NADH reoxidation, etc.) 9, 15, 16.

289

Direct fermentation of the untreated prehydrolysate had not resulted in any cell

290

growth and solvent production. In this case, detoxification treatment (overliming or

291

activated charcoal adsorption) was therefore applied to improve the fermentability of the

292

concentrated prehydrolysate. A common yet undesirable effect was that both overliming

293

and charcoal adsorption had caused the loss of carbohydrates content. Composition

294

analysis revealed that the sugar content (49.1 g/L) in the concentrated poplar pre-

295

hydrolysate decreased to 45.4/43.3 g/L following the overliming/charcoal adsorption


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Page 14 of 32

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treatment (Table 3). Similarly, overliming/activated charcoal treatment had decreased the

297

total sugar content of concentrated southern pine pre-hydrolsate from 52.4 g/L to

298

48.3/46.6 g/L. And it was primarily the oligomeric sugars rather than the monomer that

299

contributed to the carbohydrates loss.

300

Overliming treatment had increased the pH of prehydrolysate from 3.3 to 6.7,

301

which are suitable for ABE fermentation. But it had not removed any acetic acid and

302

soluble lignin. In contrast to the overliming treatment, activated charcoal adsorption had

303

substantially decreased the soluble lignin content (more than 90%) as evidenced by a

304

great reduction of the UV-Vis absorbance (Table 3). Additionally, the adsorption had

305

considerably removed the unbound acetic acid in the poplar pre-hydrolysate and reduced

306

the total acetic acid concentration to 5.4 g/L, from 9.5 g/L for the concentrated and un-

307

detoxified pre-hydrolysate. But there is no dramatic change in the pH of pre-hydrolysate

308

after charcoal treatment. Overliming or adsorption treatments affected the prehydrolysate

309

of southern pine in a similar manner as that on the poplar prehydrolysate (Table 3).

310

3.3

311

hemicellulose pre-hydrolysate

Simultaneous

saccharification

and

fermentation

of

overliming-detoxified

312

ABE fermentation with mixed sugar as feed was performed as control test (Table

313

4). As the poplar prehydrolysate control, fermentation of synthetic sugar medium (xylose,

314

35.4 g/L; glucose, 5.0 g/L; mannose, 4.7 g/L; galactose, 5.3 g/L) produced 13.6 g/L of

315

ABE solvent with 8.5 g/L of butanol over 96-h period, giving a fermentation yield of 0.27

316

(g-solvent/g-sugar) and productivity of 0.14 g/L/h (Table 4). In comparison, a total of

317

15.3 g/L of ABE solvent with 9.3 g/L of butanol was produced from a mixed sugar


14

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


318

control for the concentrated pine prehydrolysate (glucose, 5.1 g/L; xylose, 14.7 g/L;

319

galactose, 5.2 g/L; mannose, 25.1 g/L). The fermentation yield and productivity reached

320

to 0.31 (g-ABE/g-solvent) and 0.16 g/L/h.

321

Simultaneous saccharification and fermentation of detoxified prehydrolysate was

322

performed as described in section 2.6. Attempt at SSF of overliming-treated poplar pre-

323

hydrolysate was unsuccessful in that the acid (acetic, 8.3 g/L; butyric 6.6 g/L), instead of

324

the solvent (1.8 g/L), was primarily produced (so-called acid crash) with significant level

325

of unused residual sugars (10.4 g/L) as indicated in Table 4. Similarly, for overliming-

326

detoxified southern pine prehydrolysate, although 5.7 g/L of solvent was produced, the

327

acids (acetic, 6.9 g/L; butyric, 5.4 g/L) remained to be the primary products rather than

328

the solvent. The solvent yield in SSF of overliming-detoxified prehydrolysate is far less

329

than that in the mixed sugar control. It is known that ABE fermentation typically involves

330

a bi-phasic metabolic pattern, in which the acid was firstly produced in acidogenic phase

331

and then re-assimilated for solvent production in solventogenic phase. Studies have

332

proposed two possible reasons for the phenomenon of acid crash. Firstly, the buffering

333

effect (pH) of fermentation medium was disrupted due to the accumulation of

334

undissociated acid, disabling the metabolic pathway for acid re-assimilation and solvent

335

production

336

reported to interfere with the transition from acidogenesis to solventogenesis,

337

substantially reducing the solvent production 24-27.

16, 21, 23, 24

. Additionally, the presence of soluble lignin content was also

338

In contrast to the results in the present study, Sun and Liu previously reported

339

detoxification of hemicellulose hydrolysate with overliming had resulted in a ten-fold

340

increase in the solvent production from 0.8 g/L to 11 g/L


15

. In their study, prior to

15


Page 16 of 32

341

overliming treatment, nanofiltration was performed and removed a large portion of acetic

342

acid and phenolic compounds from the hydrolysate. Given the fact that overliming

343

treatment had not notably removed the acetic acid and soluble lignin content, these

344

compounds were believed to lead to the inhibition.

345

3.4 Simultaneous saccharification and fermentation of activated charcoal-detoxified

346

hemicellulose pre-hydrolysate

347

Unlike overliming, activated charcoal treatment had significantly removed the

348

phenolic compounds and partially removed the acetic acid from the hemicellulose pre-

349

hydrolysate. SSF of charcoal-treated poplar prehydrolysate produced a total of 7.6 g/L

350

ABE solvent with 5.2 g/L of butanol (Table 4). The solvent yield (0.18 g-solvent/g-sugar)

351

was substantially higher than that of overliming-treated but still lower than that of mixed

352

sugar control. At the end of fermentation, a significant level of the acetic acid (7.6 g/L)

353

and butyric acid (3.9 g/L) was present in the medium.

354

Supplementation of calcium carbonate in ABE fermentation has been reported to 16, 21

355

stabilize the pH of fermentation medium and thus, improve the solvent production

356

In the present study, SSF with the addition of CaCO3 produced a total of 10.8 g/L ABE

357

solvent (acetone, 3.5 g/L; butanol, 6.8 g/L; ethanol, 0.5 g/L), leaving insignificant level of

358

residual sugars unconsumed. Time-course profile of SSF exhibited a typical pattern of

359

ABE fermentation (Fig. 2). Within the first 24h, acetic acid and butyric acid were

360

concurrently produced, reaching to 7.6 g/L and 4.5 g/L at 24h. At the same time,

361

monomeric xylose, released from enzymatic hydrolysis, accumulated to 13.4 g/L,

362

corresponding to a 38% xylan digestibility. Afterwards, with gradual consumption of the


.

16

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363

sugars, acidogenic phase transitioned into solventogenic phase and ABE solvent was

364

produced at a significant rate. The solvent yield and productivity of SSF reached to 0.28

365

(g-solvent/g-sugar) and 0.13 g/L/h, which was comparable to that of mixed sugar control.

366

In a similar way, activated charcoal treatment had enabled a substantial

367

improvement on the solvent production for the southern pine prehydrolysate (Table 4). In

368

the acidogenic phase, monomeric mannose and xylose accumulated to 8.6 g/L and 6.1

369

g/L (Fig. 3). Although both mannose and xylose were utilized by the culture for solvent

370

production, mannose appeared to be the preferred choice as evidenced by greater

371

consumption rate than the xylose. Similar results about the sugar preference had been

372

previously reported by 9. In the end, SSF produced a total of 13.2 g/L ABE solvent

373

(butanol, 8.3 g/L; acetone, 4.2 g/L), giving a solvent yield and productivity of 0.28 (g-

374

solvent/g-sugar) and 0.14 g/L/h (Fig. 3). In this case, the yield of SSF is comparable to

375

that of pure sugar control and the addition of CaCO3 was not necessary, probably due to

376

its low acetic acid content.

377

3.4 Discussion

378

On the premise of not impairing the pulp quality and yield, integration of

379

hemicellulose pre-extraction to the traditional chemical pulping process has been

380

proposed for the forest-based biorefinery

381

210 ºC for 30-120 min) selectively removes hemicellulose from the wood while retaining

382

the cellulose portion in the solids. The resulting prehydrolysate was primarily composed

383

of xylan-oligosaccharides (hardwood) or mannan-and-xylan oligosaccharides (softwood),

384

which can be enzymatically hydrolyzed into fermentable sugars.

1, 4, 13, 14

. Hot-water extraction (typically, 150-


17


Page 18 of 32

385

The present study investigated the use of the hemicellulose prehydrolysate from

386

the hot-water treatment of two types of pulping wood as a liquid sugar source for

387

biobutanol production. A comparison of ABE production in the present study from

388

relevant studies was summarized in Table 5. Due to the presence of toxic compounds

389

(phenolic compounds) in the prehydrolysate, SSF of untreated prehydrolysate suffered

390

from severe inhibition. Activated charcoal treatment had been effective for the

391

detoxification of the hydrolysates from steam-treated corn stover 25, hydrothermo-treated

392

switchgrass 16, and dilute acid-treated eucalyptus 17. In the present study, activated carbon

393

treatment substantially removed the phenolic compounds from the hemicellulose

394

prehydrolysate and thus, significantly improved the solvent production. SSF of the

395

adsorption-treated poplar/pine pre-hydrolysate produced a total of 10.8 g/L/13.2 g/L of

396

ABE solvent, resulting in a solvent yield comparable to that of mixed sugar control.

397

Relevant studies have reported the use of green liquor extracted hardwood hydrolysate

398

(xylose, 42.7 g/L; glucose, 20.8 g/L) as feedstock for butanol production. In their study,

399

the effects of various detoxification treatments on the batch fermentation were compared

400

28

401

whereas a total of 5.8/9.0/11.4 g/L of ABE solvent was produced following the

402

overliming/activated charcoal/ion-exchange resin treatment 28. In terms of alleviating the

403

phenol-induced inhibition, it is believed that activated charcoal treatment is superior to

404

overliming. The charcoal could be regenerated by firstly desorption with 10% hydrogen

405

peroxide solution and then reactivated in 200 °C oven for overnight

406

detoxification conditions (e.g., charcoal loading and treatment time) should be further

. Fermentation of untreated hardwood prehydrolysate produced 6.7 g/L of ABE solvent


17

. However, the

18

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


407

optimized to enable a great reduction of toxic compounds but minimize the carbohydrate

408

loss.

409

4. Conclusion

410

The hemicellulose pre-hydrolysate from both the hardwood poplar and softwood

411

pine could be fermented into bio-butanol with proper detoxification treatment for the

412

reduction of phenolic compounds. Activated charcoal treatment substantially improved

413

the fermentability of the hemicellulose prehydrolysate. Fermentation of the poplar/pine-

414

derived pre-hydrolysate produced a total of 10.8/13.2 g/L ABE, corresponding to a

415

solvent yield of 0.25/0.28 (g-solvent/g-sugar), which was near to that of mixed sugar

416

control. The present study demonstrated the technical feasibility of utilizing hot-water

417

extracted prehydrolysate as feedstock for ABE production, which could be used as a

418

fundamental basis for designing a pilot-scale fermentation plant as a co-facility in the

419

pulp and paper plant.

420

421

422

423


19


424

Page 20 of 32

Acknowledgement

425

The authors would like to thank Dr. Thomas McCaskey, Department of Animal

426

Science, Auburn University, for his guidance on the culture maintenance. We gratefully

427

acknowledge the financial support from National Science Foundation (NSF) and the

428

Southeastern Regional Sun Grant Center, Knoxville, TN and AkzoNobel Pulp &

429

Performance Chemicals, Marrietta, GA. We also would like to thank Novozymes, North

430

America Inc., Franklinton, NC for providing C-Tec 2 enzyme, and Boise Paper Company,

431

Jackson, AL for providing the paper mill sludge.

432

433

434

435

436

437

438

439

440


20

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441

Reference

442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

1. Amidon, T. E.; Liu, S., Water-based woody biorefinery. Biotechnology Advances 2009, 27, (5), 542-550. 2. Helmerius, J.; von Walter, J. V.; Rova, U.; Berglund, K. A.; Hodge, D. B., Impact of hemicellulose pre-extraction for bioconversion on birch Kraft pulp properties. Bioresource Technology 2010, 101, (15), 5996-6005. 3. Yoon, S.-H.; Macewan, K.; Van Heiningen, A., Hot-water pre-extraction from loblolly pine (Pinus taeda) in an integrated forest products biorefinery. Tappi Journal 2008, 7, (6), 27-32. 4. Huang, F.; Ragauskas, A., Extraction of Hemicellulose from Loblolly Pine Woodchips and Subsequent Kraft Pulping. Industrial and Engineering Chemistry Research 2013, 52, (4), 17431749. 5. Huang, H.-J.; Ramaswamy, S.; Al-Dajani, W. W.; Tschirner, U., Process modeling and analysis of pulp mill-based integrated biorefinery with hemicellulose pre-extraction for ethanol production: A comparative study. Bioresource Technology 2010, 101, (2), 624-631. 6. Yoon, S.-H.; van Heiningen, A., Green liquor extraction of hemicelluloses from southern pine in an Integrated Forest Biorefinery. Industrial and Engineering Chemistry Research 2010, 16, (1), 74-80. 7. Duerre, P., Fermentative butanol production - Bulk chemical and biofuel. Incredible Anaerobes: from Physiology to Genomics to Fuels 2008, 1125, 353-362. 8. Green, E. M., Fermentative production of butanol - the industrial perspective. Current Opinion in Biotechnology 2011, 22, (3), 337-343. 9. Ezeji, T.; Blaschek, H. P., Fermentation of dried distillers' grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresource Technology 2008, 99, (12), 5232-5242. 10. Guan, W.; Shi, S.; Tu, M.; Lee, Y. Y., Acetone-butanol-ethanol production from Kraft paper mill sludge by simultaneous saccharification and fermentation. Bioresource Technology 2016, 200, 713-721. 11. Kudahettige-Nilsson, R. L.; Helmerius, J.; Nilsson, R. T.; Soejblom, M.; Hodge, D. B.; Rova, U., Biobutanol production by Clostridium acetobutylicum using xylose recovered from birch Kraft black liquor. Bioresource Technology 2015, 176, 71-79. 12. Sjostrom, E., CARBOHYDRATE DEGRADATION PRODUCTS FROM ALKALINE TREATMENT OF BIOMASS. Biomass and Bioenergy 1991, 1, (1), 61-64. 13. Hamaguchi, M.; Kautto, J.; Vakkilainen, E., Effects of hemicellulose extraction on the kraft pulp mill operation and energy use: Review and case study with lignin removal. Chemical Engineering Research and Design 2013, 91, (7), 1284-1291. 14. Kang, L.; Lee, Y. Y.; Yoon, S.-H.; Smith, A. J.; Krishnagopalan, G. A., ETHANOL PRODUCTION FROM THE MIXTURE OF HEMICELLULOSE PREHYDROLYSATE AND PAPER SLUDGE. BioResources 2012, 7, (3), 3607-3626. 15. Sun, Z. J.; Liu, S. J., Production of n-butanol from concentrated sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum ATCC824. Biomass and Bioenergy 2012, 39, 39-47. 16. Liu, K.; Atiyeh, H. K.; Pardo-Planas, O.; Ezeji, T. C.; Ujor, V.; Overton, J. C.; Berning, K.; Wilkins, M. R.; Tanner, R. S., Butanol production from hydrothermolysis-pretreated switchgrass: Quantification of inhibitors and detoxification of hydrolyzate. Bioresource Technology 2015, 189, 292-301.


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17. Villarreal, M. L. M.; Prata, A. M. R.; Felipe, M. G. A.; Silva, J. B. A. e., Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by Candida guilliermondii. Enzyme and Microbial Technology 2006, 40, (1), 17-24. 18. Yoon, S.-H.; Cullinan, H. T.; Krishnagopalan, G. A., Reductive Modification of Alkaline Pulping of Southern Pine, Integrated with Hydrothermal Pre-extraction of Hemicelluloses. Industrial and Engineering Chemistry Research 2010, 49, (13), 5969-5976. 19. Jun, A.; Tschirner, U. W.; Tauer, Z., Hemicellulose extraction from aspen chips prior to kraft pulping utilizing kraft white liquor. Biomass and Bioenergy 2012, 37, 229-236. 20. Zhang, Y.; Han, B.; Ezeji, T. C., Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. New biotechnology 2012, 29, (3), 345-351. 21. Yang, X.; Tu, M.; Xie, R.; Adhikari, S.; Tong, Z., A comparison of three pH control methods for revealing effects of undissociated butyric acid on specific butanol production rate in batch fermentation of Clostridium acetobutylicum. Amb Express 2013, 3. 22. Liu, S.; Lu, H.; Hu, R.; Shupe, A.; Lin, L.; Liang, B., A sustainable woody biomass biorefinery. Biotechnology Advances 2012, 30, (4), 785-810. 23. Ujor, V.; Agu, C. V.; Gopalan, V.; Ezeji, T. C., Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation. Applied Microbiology and Biotechnology 2014, 98, (14), 6511-6521. 24. Cho, D. H.; Lee, Y. J.; Um, Y.; Sang, B.-I.; Kim, Y. H., Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from Clostridium beijerinckii. Applied Microbiology and Biotechnology 2009, 83, (6), 1035-1043. 25. Wang, L.; Chen, H., Increased fermentability of enzymatically hydrolyzed steamexploded corn stover for butanol production by removal of fermentation inhibitors. Process Biochemistry 2011, 46, (2), 604-607. 26. Baral, N. R.; Shah, A., Microbial inhibitors: formation and effects on acetone-butanolethanol fermentation of lignocellulosic biomass. Applied Microbiology and Biotechnology 2014, 98, (22), 9151-9172. 27. Mechmech, F.; Chadjaa, H.; Rahni, M.; Marinova, M.; Ben Akacha, N.; Gargouri, M., Improvement of butanol production from a hardwood hemicelluloses hydrolysate by combined sugar concentration and phenols removal. Bioresource Technology 2015, 192, 287-295. 28. Lu, C.; Dong, J.; Yang, S.-T., Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process. Bioresource Technology 2013, 143, 467-475. 29. Sasaki, C.; Kushiki, Y.; Asada, C.; Nakamura, Y., Acetone-butanol-ethanol production by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) methods using acorns and wood chips of Quercus acutissima as a carbon source. Industrial Crops and Products 2014, 62, 286-292.

523 524

525

526


22

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

527


Figure Legends

528 529

Fig. 1. Effects of model inhibitors on the cell growth of C. acetobutylicum ATCC 824

530

and the detoxification effect of Tween 80

531

Fig. 2. Simultaneous saccharification and fermentation (SSF) of activated charcoal-

532

detoxified poplar pre-hydrolysate with the addition of CaCO3 with an enzyme cocktail of

533

cellulase (5 mg-protein/g-xylan) and xylanase (25 mg-protein/g-xylan)

534

Fig. 3. Simultaneous saccharification and fermentation (SSF) of activated charcoal-

535

detoxified southern pin pre-hydrolysate with an enzyme cocktail of cellulase (5 mg-

536

protein/g-xylan), xylanase (10 mg-protein/g-xylan) and pectinase (10 mg-protein/g-

537

mannan)

538

539

540

541

542

543

544

545

546

547


23


Page 24 of 32

548

549 550

Table 1

551

Chemical composition of hybrid poplar (hardwood) and southern pine (softwood) Component (%) Glucan Xylan Galactan Arabinan Mannan Lignin Ash Acetyl Solid Recovery

Hybrid Poplar Untreated Treated* 42.2±0.5 41.2±0.2 18.7±0.4 10.7±0.03 0.8±0.1 0.5±0.2 0.5±0.1 0.3±0.01 2.4±0.3 1.9±0.2 27.5±0.6 25.8±0.4 0.6 0.3 5.5±0.2 3.0±0.3 84.3

Southern Pine Untreated Treated* 40.6±0.4 39.4±0.2 6.8±0.1 4.5±0.3 2.8±0.1 1.4±0.01 1.1±0.03 0.7±0.06 9.6±0.7 5.3±0.3 32.8±0.9 30.7±0.4 0.5 0.3 2.5±0.2 1.7±0.4 86.4

552 553 554

Note. * The solid composition of hot-water treated sample is calculated on the basis of untreated raw sample.

555 556 557 558 559 560 561 562 563 564 565


24

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

566


Table 2 Chemical composition of hot-water extracted hemicellulose prehydrolyzate

567

Compone nt (g/L) Glucose Xylose Galactose Arabinose Mannose Total Acetic acid HMF Furfural Soluble lignin

Hybrid Poplar (Hardwood) Monomer Oligomer Total 0.3 1.2 1.4±0.3 2.4 14.3 16.8±0.7 0.6 0.9 1.5±0.04 0.5 0.0 0.5±0.2 0.5 1.5 2.0±0.4 4.3 17.9 22.2

Southern Pine (Softwood) Monomer Oligomer Total 0.4 1.6 2.0±0.2 1.1 3.0 4.1±0.4 0.7 2.1 2.8±0.4 1.5 0.3 1.7±0.08 0.6 8.2 8.7±0.6 4.3 15.0 19.3

2.0

-

4.8±0.4

0.7

-

1.6±0.3

0.0 0.4

-

0.0 1.3

0.1 0.1

-

0.2 0.4

3.1

-

-

3.6

-

-

568 569

Note:

570 571

a. Monomer represents the concentration of monomeric sugars and oligomer represents the concentration of oligosaccharides.

572 573

b. The value reported is UV-Vis absorbance (10x dilution) at wavelength of 290 nm, which linearly correlates with the concentration of soluble lignin content.

574 575 576


25

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Page 26 of 32

Table 3 Effects of detoxification on chemical composition of concentrated prehydrolysate Components (g/L) Glucose Xylose Galactose Arabinose Mannose Total pH Acetic acid HMF Furfural Soluble lignin

Hybrid poplar prehydrolysate Concentrated Overliming Charcoal 3.8 3.5 2.3 36.1 34.5 33.5 3.7 2.8 2.8 1.1 0.7 1.3 4.5 3.8 3.5 49.1 45.4 43.3 3.3 6.7 3.7 9.5 10.2 5.4 1.0 ND 0.1 1.8 ND 0.6 3.1 2.9 0.3

Southern pine prehydrolysate Concentrated Overliming Charcoal 5.4 5.3 4.9 12.6 10.9 11.8 7.5 7.5 6.5 2.5 2.0 2.4 24.3 22.6 20.9 52.4 48.3 46.6 3.4 6.1 3.6 3.8 3.6 2.1 1.4 ND 0.3 1.3 ND 0.7 3.6 3.4 0.4

a. ND indicates non-detectable. b. The value reported as UV-Vis absorbance (10x dilution) at wavelength of 290 nm, which linearly correlates with the concentration of soluble lignin content.


26

Page 27 of 32


1 2 3 4 Table 4 5 6 7 ABE fermentation of mixed sugars and detoxified prehydrolysate 8 9 Concentrated poplar-derived prehydrolysate Concentrated southern-pine prehydrolysate 10 Products Poplar Control Untreated CaO Carbon Carbon+CaCO3 Pine control Untreated CaO Carbon Carbon+CaCO3 11 46.6 46.6 12 Sugar (g/L) 48.3 50.3 49.1 45.4 43.3 42.7 50.1 52.4 13 Acetone (g/L) 4 4.2 4.1 0.6 2.1 3.5 5.1 1.7 14 Butanol (g/L) 7.9±0.2 8.5 1.1 5.2±0.6 6.8±0.8 9.3 3.7 8.3±0.4 15 12.6±0.6 10.8±0.5 15.3 5.7 13.2±0.4 13.6 1.8 7.6±0.7 16 ABE (g/L) 17Yield (g/g-sugar) 0.27 0.28 0.27 0.04 0.18 0.25 0.31 0.12 18 0.14 0.13 Productivity (g/L/h) 0.14 0.02 0.08 0.11 0.16 0.06 19 5.3 4.6 2.2 5.1 10.4 7.6 6.2 1.9 2.1 6.9 20Acetic acid (g/L) 21Butyric acid (g/L) 2.1 1.6 0.8 6.6 3.9 1.8 1.0 5.4 22 1.1 0.6 Rsugar (g/L) 0.8 40.8 10.4 4.6 0.9 1.2 45.5 8.1 23 24 25 Note 26 27 28 a. Synthetic medium consisted of a total of 50.3 g/L sugars, including xylose (35.4 g/L), glucose (5.0 g/L), mannose (4.7 g/L) and 29 galactose (5.3 g/L). 30 31 b. Synthetic medium consisted of a total of 50.1 g/L sugars, including glucose (5.0 g/L), xylose (14.7 g/L), mannose (25.2 g/L) and 32 galactose (5.2 g/L) 33 34 35 36 37 38 39 40 41 42 43 44 27 ACS Paragon Plus Environment 45 46 47

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 28 of 32

Table 5. Comparison of ABE production from various studies ABE (g/L) 10.8 13.2

ABE Yield (g/g sugar) 0.25 0.28

C. beijerinckii CC 101

5.8 9.0 11.4

0.28 0.28 0.39

Nanofiltration+overliming

C. acetobutylicum ATCC 824

11.0

0.28

15

Kraft pulping

Activated charcoal


2.8

0.12

11

Hardwood pre-hydrolysate

Hot-water

Flocculation Nanofiltration/flocculation


6.4 4.3

0.17 NA

Acorns hydrolysate

Steamexplosion

Two-stage extraction

C. acetobutylicum NBRC 13948

15.3

NA

Switchgrass

Hydrothermo

None pH adjustment (NaOH) NaOH+CaCO3 Adsorption


1.0 5.6 7.9 16.8

0.12 0.16 0. 0.30

Corn stover

Steamexplosion

Activated charcoal


12.4

0.30

Feedstocks

Pretreatment

Detoxification

Culture

Poplar pre-hydrolysate Pine pre-hydrolysate

Hot-water

Activated charcoal


Mixed wood pre-hydrolysate

Green liquor

Overliming Activated charcoal Ion-exchange resin

Maple pre-hydrolysate

Hot-water

Xylan in black liquor


Reference This study

28

27

29

16

25

28

Page 29 of 32

2.0

2.0

Ferulic acid

1.5

Optical Density (OD) at 600 nm


p-Coumaric acid

1.0

0.5

0.0

1.5

1.0

0.5

0.0 0.0

0.5

1.0

1.5

2.0

0.0

0.5

Dosage (g/L)

1.0

1.5

2.0

Dosage (g/L)

2.0

2.0

Vanillic acid

4-Hydroxybenzoic acid

1.5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

0.5

0.0

1.5

1.0

0.5

0.0 0.0

0.5

1.0

1.5

2.0

0.0

Dosage (g/L)

0.5

1.0

1.5

2.0

Dosage (g/L)

Fig. 1 Effects of model inhibitors on the cell growth of C. acetobutylicum ATCC 824 and the detoxification effect of Tween 80


29


Page 30 of 32

Fig. 2 Simultaneous saccharification and fermentation (SSF) of activated charcoaldetoxified poplar pre-hydrolysate with the addition of CaCO3 with an enzyme cocktail of cellulase (5 mg-protein/g-xylan) and xylanase (25 mg-protein/g-xylan)


30

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Fig. 3 Simultaneous saccharification and fermentation (SSF) of activated charcoaldetoxified southern pin pre-hydrolysate with an enzyme cocktail of cellulase (5 mgprotein/g-xylan), xylanase (10 mg-protein/g-xylan) and pectinase (10 mg-protein/gmannan)


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Acetone-butanol-ethanol (ABE) production from fermentation of hot

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