Bioresource Technology

Improving the biogas yield of manure: Effect of Pretreatment on Anaerobic Digestion of

the recalcitrant fraction of manure
--Manuscript Draft--

Manuscript Number: BITE-D-20-07453R2

Article Type: Original research paper

Keywords: anaerobic digestion; methane yield; Digested Manure Fibers; Variable Pretreatment
conditions; Composition analysis

Corresponding Author: Birgitte K Ahring, PhD

Washington State University-Tricities
Richland, Washington UNITED STATES

First Author: Muhammad Usman khan, PhD

Order of Authors: Muhammad Usman khan, PhD

Birgitte K Ahring, PhD

Abstract: Anaerobic digestion of animal manure show a maximum of ca. 50% conversion of
volatile solids due to recalcitrance of lignin and crystalline cellulose under anaerobic
conditions.  The aim of this study is to evaluate different pretreatments on anaerobic
digestion of manure fibers after anaerobic digestion. Physical, chemical, thermal, and
thermal combined with alkaline pretreatments using sodium hydroxide were tested on
manure fiber fraction separated out after anaerobic digestion of cow manure. The
anaerobic digestion after pretreatment along with untreated controls were done in
semi-continuous anaerobic bioreactors. All pretreatments showed positive effect and
the highest increase in VS conversion (42.4%) and methane yield (ca. 127%) was
found for 3% NaOH pretreated sample at 180oC. Composition analysis showed that
cellulose, hemicellulose and lignin in digested manure fibers were optimally reduced by
24.8, 29.1 and 9.5% respectively during pretreatment and 76.5% of cellulose and
84.9% of hemicellulose was converted to methane during AD

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Cover Letter

November 13, 2020

Dear Editor Dr Christian Larroche

We greatly appreciate your and reviewers’ time and helps with this manuscript! Attached please find a MS
Word file of our revised manuscript (BITE-D-20-07453) “Improving the biogas yield of manure: Effect
of Pretreatment on AD of the recalcitrant fraction of manure” which we would like to publish in Journal
of Bioresource Technology. We revised the manuscript according to the reviewers and editors’ comments
with a clean version of manuscript, changes marked manuscript, and also outlined each change made (point
by point) with Response to Reviewers. Thank you very much for your time and considerations! Please feel
free to contact me if you have any questions.

Sincerely,

Birgitte K. Ahring, Ph.D.

Battelle Distinguished Professor

Bioproducts, Sciences and Engineering Laboratory, BSEL

2710 Crimson Way, Richland, WA 99354-1671

509.372.7625 | www.tricities.wsu.edu
Detailed Response to Reviewers

November 13, 2019

To:
Bioresource Technology
Editor Dr. Christian Larroche,

Dear Dr. Pandey,

Enclosed please find our revised manuscript (BITE-D-20-07453) “Improving the biogas yield of
manure: Effect of Pretreatment on Anaerobic Digestion of the recalcitrant fraction of
manure” along with a marked-up version of the original manuscript showing the parts, which have
been changed due to the collected comments of the reviewers:

The following revisions have been done:

-Editor's note:

Comment 1
- Each highlight can be maximum 85 characters including spaces?

Our response: Highlights have been revised keeping in view the criteria of the journal.

Comment 2
- Avoid full justification of the text?

Our response: The appropriate changes have been made in the revised manuscript.

Comment 3
- Number references in the list and keep in mind that they cannot be more than 50?

Our response: The number of reference have been reduced to 49 in the revised manuscript to
meet the criteria of the journal.
Yours sincerely,

Birgitte K. Ahring, Ph.D.

Professor, Head of BioScience and Technology Group
Bioproduct Sciences and Engineering Laboratory, BSEL
Washington State University
2710 Crimson Way
Richland, WA 99354-1671
Phone: 509-372-7682
Fax: 509-372-7690
Email: bka@wsu.edu
Highlights (for review)

Highlights

 Effect of different pretreatments on AD of manure fibers was investigated

 Alkali assisted thermal pretreatment improved degradation of manure fibers
 Methane Yield improved by 127% by thermal pretreatment with 3% NaOH added
 Cellulose, hemicellulose and VS was also highest with 3% NaOH pretreatment
Detailed Response
Highlights (for review)
to Reviewers

November 13, 2019

To:
Bioresource Technology
Editor Dr. Christian Larroche,

Dear Dr. Pandey,

Enclosed please find our revised manuscript (BITE-D-20-07453) “Improving the biogas yield of
manure: Effect of Pretreatment on Anaerobic Digestion of the recalcitrant fraction of
manure” along with a marked-up version of the original manuscript showing the parts, which have
been changed due to the collected comments of the reviewers:

The following revisions have been done:

-Editor's note:

Comment 1
- Each highlight can be maximum 85 characters including spaces?

Our response: Highlights have been revised keeping in view the criteria of the journal.

Comment 2
- Avoid full justification of the text?

Our response: The appropriate changes have been made in the revised manuscript.

Comment 3
- Number references in the list and keep in mind that they cannot be more than 50?

Our response: The number of reference have been reduced to 49 in the revised manuscript to
meet the criteria of the journal.
Yours sincerely,

Birgitte K. Ahring, Ph.D.

Professor, Head of BioScience and Technology Group
Bioproduct Sciences and Engineering Laboratory, BSEL
Washington State University
2710 Crimson Way
Richland, WA 99354-1671
Phone: 509-372-7682
Fax: 509-372-7690
Email: bka@wsu.edu
Manuscript Click here to view linked References

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4 Improving the biogas yield of manure: Effect of Pretreatment on Anaerobic Digestion of
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6 the recalcitrant fraction of manure.
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8 Muhammad Usman Khana,c, Birgitte Kiaer Ahringa,b,c*
9 a
10 Bioproducts, Sciences and Engineering Laboratory, Washington State University, Tri-Cities,
11 Richland, WA-99354, bGene and Linda Voiland School of Chemical Engineering and
12 Bioengineering, cBiological Systems Engineering, Washington State University, Pullman, WA-
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42 Corresponding author
43 Dr. Birgitte K. Ahring
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45 Bioproducts Sciences and Engineering Laboratory
46 Washington State University, Tri-cities
47 2710 Crimson Way
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Richland, WA 99354
50 Tel.: 01-(509)-372-7682
51 Fax: 01-(509)-372-7690.
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E-mail address: bka@wsu.edu
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4 Abstract
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7 Anaerobic digestion of animal manure show a maximum of ca. 50% conversion of volatile solids
8
9 due to recalcitrance of lignin and crystalline cellulose under anaerobic conditions. The aim of this
10
11 study is to evaluate different pretreatments on anaerobic digestion of manure fibers after anaerobic
12 digestion. Physical, chemical, thermal, and thermal combined with alkaline pretreatments using
13
14 sodium hydroxide were tested on manure fiber fraction separated out after anaerobic digestion of
15
16 cow manure. The anaerobic digestion after pretreatment along with untreated controls were done
17
18 in semi-continuous anaerobic bioreactors. All pretreatments showed positive effect and the highest
19
20 increase in VS conversion (42.4%) and methane yield (ca. 127%) was found for 3% NaOH
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pretreated sample at 180oC. Composition analysis showed that cellulose, hemicellulose and lignin
23 in digested manure fibers were optimally reduced by 24.8, 29.1 and 9.5% respectively during
24
25 pretreatment and 76.5% of cellulose and 84.9% of hemicellulose was converted to methane during
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27 AD.
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30 Key Words
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32 Anaerobic digestion; Methane yield; Digested Manure Fibers; Variable Pretreatment conditions;
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34 Composition analysis
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7 1. Introduction
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9 Anaerobic digestion is a promising renewable energy technology allowing for waste reduction
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11 while producing renewable energy and a digestate high in nutrients such as nitrogen and
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13 phosphorous. The anaerobic digestion has, therefore, environmental benefits such as size reduction
14 of the waste material, minimizing the emission of hazardous greenhouse gases, production of
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16 organic fertilizer, reduction of odor from the waste material besides production of renewable
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18 methane. Anaerobic digestion is, therefore, considered to be viable option for managing large
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20 amount of waste products (Igoni et al., 2008). The methane gas produced by anaerobic digestion
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22 can be used for heating, cooking, as a fuel for transport or as a green bio-natural gas for addition
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24 to the natural gas grid (Pöschl et al., 2010). All use of biogas produced from anaerobic digestion
25 will be an alternative to fossil fuels, which will lower the greenhouse gas emission to the
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27 atmosphere (Leet et al., 2020).
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30 Generally, municipal solid waste, manure, sewage sludge and organic waste from agricultural and
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32 industrial activities are used as a feedstock for biogas plants (Hamelin et al., 2008). Besides these
33 materials lignocellulosic biomass materials such as straw and husks are being considered for
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35 production of methane due to the abundance of these materials (Khan and Ahring, 2020).
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37 However, anaerobic digestion of these materials is faced by different challenges. Firstly, these
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39 materials are recalcitrant and only 20-30% of the volatile solids will be converted to biogas during
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41 anaerobic digestion (Kucharska et al., 2018, Sawatdeenarunat et al., 2018, Surendra et al.,
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2018a, Surendra et al., 2018b). Secondly, the need for anaerobic reactor systems, which can
44 handle feed stocks with high amounts of fibers, is not the conventional systems, which is
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46 implemented right now. Thirdly, there is a need for develoment of storage systems for storing
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48 digested residue and effluent after digestion of lignocellulosic materials (Sawatdeenarunat et al.,
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50 2015). Finding ways to make lignocellulosic materials accessible for anaerobic digestion is,
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52 therefore, of major importance for converting lignocellulosic materials under anaerobic conditions.
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54 Some major advances in anaerobic digestion technology have been gained over the last years such
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56 as better resource recovery, understanding of the microbiome of anaerobic digestion, upgrading of
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58 biogas to natural gas quality, application of biochar in AD systems, control of AD systems using
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4 neural network technologies, utilization and management of digested waste materials as well as
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6 application of 3D printing technology within the bioreactor technology (Khanal et al., 2020).
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9 Lignocellulosic material is an essential component of animal manure and constitutes about 40 to
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11 60% of the manure. As a result manure is only partly converted to biogas during AD (Khan and
12 Ahring, 2020). Therefore, technologies are needed to improve the efficiency of anaerobic digestion
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14 of lignocellulosesic materials. Pretreatment of the lignocellulosic part of manure, which is left
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16 unattended after AD has been shown to improve the biogas yield of AD (Abraham et al., 2020).
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18 After pretreatment lignocellulosic materials give a high biogas yield and can substitute the need
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20 for addition organic industrial waste to boost the methane production and make the biogas plant
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economically viable (Zhao et al., 2018).
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24 Manure fibers is the solid fraction of the manure, which can be obtained by separating out the
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26 manure liquid. The fraction obtained after AD is especially interesting as it contains the recalcitrant
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28 lignocellulosic part of manure (Liao et al., 2010). There are several studies showing the potential
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30 of these manure fibers as a good lignocellulosic feed stock for biofuels production including biogas
31 (Mirtsou-Xanthopoulou et al., 2019, Khan and Ahring, 2020). Using manure fibers as feed material
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33 for biogas plants has the advantage that they are already collected and available at the biogas plant.
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35 The anaerobic digestion of manure fibers after pretreatment further makes more of the nutrients in
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37 this fraction available as a fertilizer.
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39 The fibrous part of manure consist of fibers from lignocellulosic materials used as feed for the
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41 animals along with soil, skin and hair from roughage (Jurado et al., 2013). The main challenge for
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43 anaerobic digestion of this part is their low digestibility due to the higher concentrations of
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45 lignocellulosic materials, which is limiting methane production and making the amount dependent
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47 on the retention time of the fibers in the biogas reactor. Today, many large-scale biogas plants are
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operating with a long retention time (up to 60 days) to allow for digestion of a larger fraction of
50 these fibers. Lignin is one of the major components of this material, where it will acts as a glue
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52 covering the carbohydrate fraction. The primary aim of pretreatment is to disrupt the
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54 lignocellulosic matrix to improve hydrolysis of the cellulose and hemicellulose polymers and
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56 potentially further parts of the lignin (Biswas et al., 2015). Due to the shielding of the cellulose
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58 fraction of lignocellulosic material by lignin, pretreatment is necessary to convert major parts of
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4 the cellulose, which is the biggest potential for extra methane of manure fibers (Khan and Ahring,
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6 2019).
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9 Previously, different pretreatments have been examined for improving anaerobic digestion of the
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11 recalcitrant part of manure. Tsapekos et al., 2016 studied the effect of different physical and
12 thermal alkaline pretreatments on anaerobic digestion of digested manure fibers. The results of
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14 study revealed that the pretreatment with 6% NaOH at 55 °C showed better degradation compared
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16 to other chemical and physical pretreatments and resulted in a 26% improvement of the methane
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18 yield. Bruni et al., 2010 studied the effect of size reduction on the methane productivity of
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20 undigested manure fibers and the results of the study indicated that size reduction had a positive
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effect on the methane productivity of manure fibers with 8% increase in methane yield. Angelidaki
23 and Ahring, 2000 pretreated the digested manure fibers using NH4OH. The degradation of the
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25 pretreated manure fibers improved as a result of NH4OH pretreatment resulting in a 20% increase
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27 in methane yield. Mirtsou-Xanthopoulou et al., 2019 studied the effect of aqueous ammonia
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29 soaking pretreatment on the anaerobic digestion of digested manure fibers under mesophilic (38
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31 °C) conditions using a CSTR-type anaerobic reactors. The results of that study indicated that the
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aqueous ammonia soaking pretreatment had a profound effect on the hydrolysis of carbohydrates
34 resulted in 49-68% increase in methane yield. Biswas et al., 2012 used Wet explosion (WEx)
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36 pretreatment under five different conditions to pretreat the digested manure fibers. The manure
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38 fibers pretreated at 180oC for 10 minutes showed the highest methane potential with 136% increase
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40 compared to the non-pretreated manure fibers. Jurado et al., 2013 studied the effect of aqueous
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42 ammonia soaking (AAS) pretreatment on the anaerobic digestion of digested and raw manure
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fibers. The results of the study showed that the aqueous ammonia soaking (AAS) pretreatment at
45 22 °C for 3 days resulted in from 30 to 178% improvements of the methane yield of digested and
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47 raw manure fibers, respectively.
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50 Currently, different physical, thermal, chemical, thermochemical, ultrasonic and biological
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pretreatments are being used for anaerobic digestion of lignocellolic materials. Among all these
53 pretreatments alkali pretreatments has the advantages of a better solubilization of the lignin
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55 fraction of biomass material resulting in an improved efficiency of AD process (Abraham et al.,
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57 2020). During alkali pretreatment of lignocellulosic materials solvation and saponification reaction
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59 further occur, which increases their surface area and hence the enzymatic hydrolysis of the material
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4 during AD allowing for better access by the anaerobic bacteria (Kim et al., 2016). Besides the
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6 lignin solubilized due to the breakage of ester bonds by saponification reactions (Kim et al., 2016).
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8 Thermal alkaline pretreatment of biomass causes de-crystallization and degradation of
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10 hemicellulose and cellulose (Dasgupta and Chandel, 2020). Calcium hydroxide (Ca(OH)2),
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12 sodium hydroxide (NaOH), ammonium hydroxide (NH4OH), potassium hydroxide (KOH) and
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ammonia (NH3) are the commonly used alkalis for alkali pretreatment of lignocellulosic materials.
15 Among all of these chemicals NaOH is of prime importance due to its effectiveness as well as
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17 easier handling compared to powdery materials (Taherzadeh and Karimi, 2008). However, NaOH
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19 pretreatment at room temperature requires significant time and high alkali additions to be effective
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21 (Pang et al., 2008). Moreover, the higher alkali concentrations during pretreatment cause an
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23 increase of Na+ ions in the pretreated material, which is not only inhibitory for anaerobic bacteria
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but further changes the quality of the digestate for soil amendment (Solé-Bundó et al., 2017).
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27 In all of the above studies it is obvious that pretreatment has increased the digestibility of the
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29 manure fibers. However, these studies was mainly done at lower temperatures from room
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31 temperatures to temperatures at maximum 120oC. Operating at higher temperatures might on the
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other hand be far more efficient and allow for less addition of alkali to perform the same function.
34 In present study we have examined the effect of pretreatments at room temperature as well as at
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36 higher temperature (180oC) with and without addition of NaOH. To our best knowledge this study
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38 is the first time that physical, chemical, thermal and thermochemical pretreatments at higher
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40 temperature (ca. 180oC) is compared while using lower concentrations of alkali addition. The
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42 effect of each pretreatment on the composition of the lignocellulosic polymers as well the
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degradation of these polymers during anaerobic digestion is further studied for the first time in this
45 paper.
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48 This study aims at: (1) compare the effects of different pretreatments on composition of digested
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50 manure fibers, (2) to analyze the effect of pretreatment on the compositional analysis of the manure
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fibers before and after anaerobic digestion, (3) to produce and understanding of the pretreatment
53 conditions, which allow for the maximum digestibility of the manure under anaerobic digestion.
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56 2. Material and Methods
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58 2.1. Feed Stock and Inoculum
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4 Samples for this study were collected from George DeRuyter and Sons Dairy Farm in Outlook,
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6 WA. The manure used at this plant is mixed with water to allow for use of a flush handling system
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8 to move manure from the stable to biogas system. The dilute manure waste water is passed through
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10 a clarifier before entering the biogas plant. A plug flow anaerobic digester is used for the anaerobic
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12 digestion of the manure under mesophilic conditions using a hydraulic retention time of 20 days.
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The digested manure from the digester is then pumped to a slope screen connected with a
15 dewatering roller system. The digested manure is pressed in the dewatering roller, collecting the
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17 water in one fraction and manure fibers in another fraction with an approximate moisture content
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19 of 72%. Representative samples of manure fibers and digested manure water were collected and
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21 transferred to Washington State University (WSU) Tri-cities.
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23 The inoculum for the experiments came from a 2 liters biogas reactor operated in the laboratory
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25 on cow manure at thermophilic conditions (53oC) using a hydraulic retention time of 20 days.
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28 A total of seven bio-reactors used for the testing were filled with 1L digested manure while using
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30 a N2/CO2 (80:20) gas mixture to ensure anaerobic conditions during start-up. All the reactors were
31 fed daily with the digested manure at a retention time of 20 days until the gas production was stable
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33 and similar in all of the reactors.
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36 2.2. Pretreatment of the Manure Fibers
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38 The TS contents of the manure fibers were adjusted to 10% by adding deionized water and then
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40 the particle size of the fibers was reduced to 3 mm using a lab scale blender. The materials were
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42 pretreated at 180oC using 2L Parr reactor under four different conditions without any chemical,
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44 and with NaOH (1-3% w/w) as shown in Table 1. After 30 minutes of pretreatment, the Parr reactor
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46 was cooled instantaneously using ice. The pretreated material was removed from reactor and stored
47 at 4oC. The thermal pretreatment was carried out without adding chemicals and the chemical
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49 pretreatment was carried out using 2% NaOH for 30 minutes at room temperature. The control
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51 sample used for this study did not undergo any pretreatment. All the pretreatment conditions,
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53 NaOH concentrations, temperature, time and the total solids content during pretreatment has been
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55 presented in Table 1.
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57 2.3. Semi Continuous Anaerobic Digestion
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4 The pretreated and non-pretreated manure fibers were mixed with digested manure water to adjust
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6 the TS of all the samples to 6% and the material was stored at 4oC to avoid microbial degradation.
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8 Anaerobic digestion of pretreated and untreated samples was carried out in seven 2L bio reactors
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10 (R1-R7). The reactor R1 was used as a control and fed with untreated manure fibers, reactor R2
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12 was fed by physically pretreated manure fibers, reactor R3 was fed by manure fibers with chemical
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pretreatment alone, reactor R4 was fed by manure fibers undergone thermal pretreatment alone,
15 reactor R5 was fed by manure fibers pretreated at 180oC with addition of 1% NaOH, reactor R6
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17 was fed by manure fibers pretreated at 180oC with addition of 2% NaOH and reactor R7 was fed
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19 by manure fibers pretreated at 180oC with addition of 3% NaOH as catalyst. All the reactors were
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21 operated under thermophilic conditions at 53oC using a hydraulic retention time of 20 days and
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23 gas production from all the reactors was measured daily. The reactors were fed on a daily basis
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and the effluent samples were stored at 4oC for further analysis.
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27 2.4. Feedstock and Effluent characterization
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30 National renewable energy laboratory standards NREL standard methods were used to measure
31 the total solids (TS) content of the feed and effluent samples (Sluiter et al., 2008). The samples
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33 were dried in an oven at 105±3oC for 24h, after which it was removed and cooled to room
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35 temperature before weighing. This samples was then heated to 575 ± 25 °C in a muffle furnace
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37 according to the method described by (Sluiter et al., 2008). After 6 hours the samples were taken
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39 out from the muffle furnace and cooled to room temperature overnight in a desiccator before
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determining the Volatile solids (VS) as the lost weight of the samples.
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43 2.5. Composition analysis
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46 The lignin and carbohydrates contents of the feed and effluent samples were analyzed by drying
47 the samples at room temperature. The dried sample was then used for the analysis of lignin
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49 (Soluble and Insoluble) and carbohydrates (Cellulose and Hemicellulose) content according to the
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51 method described by Hames et al., 2008 and Sluiter et al., 2008. The Insoluble lignin and
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53 carbohydrates content of the samples were analyzed after hydrolysis and the soluble lignin content
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55 of the samples were determined using a UV spectrophotometer (Jenway 6405 UV/Visible NJ,
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57 USA). Carbohydrates contents of samples was analyzed using UltiMate® 3000 HPLC system
58 (Dionex, Sunnyvale, CA) with an Aminex® 87H Column 250 × 4.6 mm (Bio-Rad, Hercules, CA)
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60 and a Shodex RI-101 refractive index detector at 60 °C using 4 mM H2SO4 as an eluent with a
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4 flow rate of 0.6 ml/min. The compositional analysis of feed and effluent samples in triplicates and
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6 average of all the results was used as final results.
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9 2.6 Statistical Analysis
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11 The effect of pretreatment on methane yield was analyzed by one way ANOVA test and the effect
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13 of different pretreatments on degradation of cellulose, hemicellulose and volatile solids was
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15 analyzed by two way ANOVA test using GraphPad Prism V8.
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17
18
3. Results and Discussion
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20
3.1. Feed stock and Effluent Characteristics
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The composition of the digested manure fibers before pretreatment, after pretreatment and after
23 anaerobic digestion are presented in Table 2. It is evident from the experimental table that cellulose
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25 is the main fraction of untreated digested manure fibers (37.1%) followed by lignin (36.7%) while
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27 hemicellulose makes up the least abundant polymer of 26.1%. The compositional assessment of
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29 the digested manure fibers reveal that the fibers are recalcitrant to further biological degradation
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31 in its original form due to the high concentration of lignin. Moreover crystalline cellulose is also
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33
difficult to degrade. Moreover, upon analyzing the datasets from Table 2, we found that the
34 percentage of all polymers including cellulose, hemicellulose and lignin decreased in all reactor
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36 receiving pretreated materials. The reduction of cellulose, hemicellulose and lignin during all types
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38 of pretreatments is shown in Fig 1. The highest reduction of cellulose, hemicellulose and lignin
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40 were 24.8, 29.1 and 9.5%, respectively, in the sample pretreated at 180oC with the highest dose of
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42 NaOH (3%) before the digestion. The results of compositional analysis of the manure fibers after
43 pretreatments are comparable with Zhang et al., 2013. The cellulose reduction in our study is,
44
45 however, higher which probably is due to the fact that the pretreatment was carried out a higher
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47 temperature in our study, which previously have been found to significantly increase the cellulose
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49 degradation (Biswas et al., 2014). Cellulose is a crystalline polymer protected by lignin and
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51 pretreatment is essential for its conversion to biofuel. The alkali pretreatment had been found
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53 previously to alter the crystallinity of lignocellulosic materials. The cations of alkalis such as
54 sodium, potassium and calcium reacts with cellulose and cause its swelling. The swelling of
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56 cellulose reduces its crystallinity and makes it more degradable. Among all the alkalis used for
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58 pretreatment of lignocellulosic materials sodium had been found to cause highest swelling and
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60 sodium can increase the swelling of cellulose by 3 times (Karimi et al., 2013). The highest
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4 reduction was seen for the hemicellulose content probably due to the higher degradability of this
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6 polymer compared to lignin and cellulose. Hemicellulose can further react easily with NaOH
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8 compared to the other polymers which may also be a reason of its higher degradation during
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10 pretreatment (Zhang et al., 2013). Hemicellulose is an amorphous polymer and it is widely known
11
12 that this fraction of lignocellulosic materials is affected most during alkali pretreatment (Pal et al.,
13
14
2002). During alkali pretreatment of lignocellulosic materials the action of hydroxyl ion (OH-) of
15 alkalis cause swelling of cellulose and breakage of hydrogen bonds between cellulose and
16
17 hemicellulose. Moreover ester bonds between polysaccharides of lignocellulosic materials
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19 hydrolyzed, which causes solubilization of hemicellulose (Sun and Sun, 2002). This solubilization
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21 may also cause the higher reduction of hemicellulose during pretreatment process. Pretreatment of
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23 lignocellulosic materials at elevated temperature will further cause hydrolyzation of acetyl groups
24
25
of hemicellulose. This results in higher concentrations of acetic acid, which helps in auto
26 hydrolysis of lignocellulosic materials (Biswas et al., 2020). Besides this, the uronic acid
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28 substituents of hemicellulose may also remove during NaOH pretreatment. The removal of acetyl
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30 groups and uronic acid substituents improved its biodegradability as they are known to hinder the
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32 enzyme accessibility (Shetty et al., 2017). Xiao et al. (2001) observed that the hemicellulose
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34 reduction during pretreatment increased with increasing concentration of NaOH. The decrease in
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cellulose, hemicellulose and lignin contents during pretreatment may be due to conversion of
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37 insoluble fibers into soluble fragments, which can be digested easily during the downstream
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39 anaerobic digestion process (Pang et al., 2008). Cellulose and hemicellulose polymers of
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41 lignocellulosic materials are protected by lignin fraction, which acts as a shield during enzymatic
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43 hydrolysis (Taherzadeh and Karimi, 2008). Lignin is an aromatic polymer and it resist the
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45 accessibility of bacteria to carbohydrate component of the material. Moreover, lignin is difficult
46 to degrade compared to cellulose and hemicellulose fraction of lignocellulosic materials.
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48 Therefore, lignin removal by pretreatment may improve the accessibility of hydrolytic anaerobic
49
50 bacteria to carbohydrate fraction of biomass. In our study the lignin reduction during pretreatment
51
52 was found to be lower than the other polymers and it was also observed that significant lignin
53
54 reduction was only found with alkali assisted thermal pretreatments. The results of lignin reduction
55
56 during pretreatments are comparable with Karimi et al. (2013), who observed that lignin removal
57 only occur during alkali assisted NaOH pretreatment. The lignin contents of pretreated manure
58
59 fibers reduced after thermal pretreatments with NaOH and the lignin reduction rate increase with
60
61
62 10
63
64
65
 1
2
3
4 increasing concentrations of NaOH during the pretreatment. Similar results were observed
5
6 previously by Jiang et al. (2020). The decrease in lignin contents during alkali pretreatment is
7
8 probably due to the cleavage of the ether bonds (R1-O-R1) of lignin during alkali pretreatment.
9
10 Moreover, phenolic hydroxyl groups of lignin has also been observed to be affected by alkali
11
12 pretreatment which may also reduce the lignin concentration during pretreatment (Lai et al., 2019).
13
14
The overall effect of NaOH thermal pretreatment was not only by dissolution of parts of the lignin
15 but also by exposing the carbohydrates fraction allowing for extensive biodegradation of this
16
17 fraction. In accordance the cellulose and hemicellulose content decreased in all the effluent
18
19 samples (Table 2). The lignin content increased in the effluent of all the bioreactors treating manure
20
21 fibers after pretreatment showing that the cellulose and hemicellulose were the main polymers
22
23 found in the manure fibers, which were converted to methane during anaerobic digestion process,
24
25
while a larger part of the lignin still showed some resistance to biodegradation. The highest
26 reduction of the carbohydrates was found for the effluent sample with 3% NaOH followed by 2%
27
28 and 1% NaOH indicating that the degradation of cellulose and hemicellulose increased with
29
30 increasing concentrations of NaOH.
31
32
33
3.2. Semi-continues anaerobic digestion
34
35
The gas yield from all the seven reactors fed with differently pretreated materials are presented in
36 Fig 2. For the first 30 days all the reactors were operated on digested manure. Once the gas yield
37
38 become steady and similar in all the reactors, feeding with digested manure fibers was started.
39
40 Reactor R1 was fed with untreated manure fibers; R2 with physically pretreated manure fibers;
41
42 R3 with pretreated manure fibers with base (2% NaOH) addition without heat; R4 with thermally
43
44 pretreated manure fibers; R5 with thermochemical pretreated manure fibers with 1% NaOH; R6
45
46
with thermochemical pretreated manure fibers with 2% NaOH; R7 with thermochemical pretreated
47 manure fibers with 3% NaOH. The untreated and pretreated manure fibers were anaerobically
48
49 digested for 60 days with 20 days retention time (3 HRT’s) and the gas yield was monitored
50
51 continuously. Total biogas, total methane and the average methane yield of each of the samples
52
53 were calculated to evaluate the effect of pretreatment on energy generation. The total biogas and
54
55 methane yield were calculated by adding the daily yield from the day when all the reactors were
56
started feeding with manure fibers until the end of experiment. The methane yield was calculated
57
58 by dividing the average methane yield by the VS contents of the feed material. The results of total
59
60 biogas, total methane and average methane yield (ml/gVS) are presented in Fig 3. The results
61
62 11
63
64
65
 1
2
3
4 showed that all pretreatments have a positive effect on anaerobic digestion of the digested manure
5
6 fibers. Combined Thermochemical pretreatment with 3% NaOH was found to be give the highest
7
8 methane yield, while the control reactor fed with untreated manure fibers showed the lowest
9
10 methane yield. The average, maximum and minimum methane yield from R1 to R6 are presented
11
12 in Fig 4.
13
14 The methane yield is effected significantly by each type of pretreatment (P < 0.05) as shown in
15
16 Table 2. Overall the methane yield increased by 8.8, 11.1, 47.5, 86, 110.4 and 127.1% by physical,
17
18 chemical, thermal, thermochemical pretreatment with 1% NaOH, thermochemical pretreatment
19
20 with 2%NaOH and thermochemical pretreatment with 3% NaOH, respectively. Overall the 3%
21
22
NaOH pretreated samples yielded the highest methane compared to other samples which may be
23 due to the increase of soluble organic matter concentration during pretreatment, which can be
24
25 degraded easily during anaerobic digestion. Besides this the swelling of manure fibers could
26
27 physically be seen in the samples with NaOH pretreatment, which enlarged the surface area of the
28
29 material. This might have increased its microbial conversion and methane production in these
30
31 samples as previously described by Lin et al., 2009.
32
33 The increase in methane yield by physical pretreatment of manure fibers are comparable with
34
35 Bruni et al., 2010 who observed an 8% increase in methane yield by physical pretreatment. The
36
37 results of methane yield improvement by chemical pretreatments are comparable with Angelidaki
38
39 and Ahring, 2000 who observed a methane yield improvement of 13% by chemical pretreatment.
40
41
The methane yield by chemical pretreatment alone is lower in our study compared to Angelidaki
42 and Ahring, 2000 probably due to a shorter time reaction time compared to the previous study.
43
44 The results of thermal pretreatment are comparable with Mladenovska et al., 2006 who observed
45
46 24% increase in methane yield by thermal pretreatment. The higher methane yield by thermal
47
48 pretreatment in our study compared to Mladenovska et al., 2006 is probably due to the higher
49
50 pretreatment temperature. The results of thermochemical pretreatments are comparable with Jurad
51
52
et al., 2013 who observed a methane yield increase of 139% and 178% by thermochemical
53 pretreatment. Specifically we found that the methane yield improved by 127.1% by combined
54
55 NaOH addition and thermochemical pretreatment.
56
57
58
59
60 3.3. Degradation of the manure Fibers
61
62 12
63
64
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 1
2
3
4 The efficiency of semi-continuous anaerobic digestion of manure fibers after different
5
6 pretreatments were evaluated by biogas production, composition analysis and VS reduction of the
7
8 samples. The degradation of cellulose, hemicellulose and volatile solids are presented in Fig 5. It
9
10 can be seen from this figure that VS reduction as well as polymer degradation are lowest in the
11
12 control samples without any pretreatment. The degradation of the polymers in the manure fibers
13
14
as well as VS increased with increasingly severe pretreatment processes as shown in Fig 1 during
15 AD of pretreated manure fibers. The highest cellulose and hemicellulose degradation was observed
16
17 in the samples, which was pretreated with 3% NaOH where 76.5% of the cellulose, 84.9% of the
18
19 hemicellulose and 42.4% of volatile solids of the manure fibers were removed during the anaerobic
20
21 digestion process. The cellulose, hemicellulose and VS degradation in control sample was 51%,
22
23 54.2% and 14.5%, respectively. The highest cellulose, hemicellulose and VS reduction are in
24
25
agreement with the result from the measurement of the methane yield of the samples. The results
26 of the biodegradability of the manure fibers are comparable with Tsapekos et al., 2016, who
27
28 observed that the pretreatment of digested manure fibers at 55oC with 6% NaOH resulted in 80.5%
29
30 of the cellulose, 85.5% of the hemicellulose and 45% of the volatile solids were converted into
31
32 methane during the anaerobic digestion process.
33
34 The higher cellulose degradation with NaOH added during thermal pretreatment compared to the
35
36 control sample with only thermal pretreatment might be due to a lower crystallinity of cellulose
37
38 with alkali addition (Karimi et al., 2013). Among all the factors affecting the digestibility of
39
40 lignocellulosic materials such as delignification, cellulose crystallinity, hemicellulose and lignin
41
42 contents of material and hydrogen bonds, cellulose accessibility is considered to be most important
43
44
factor (Huang at al., 2010). The lower cellulose degradation in control sample may be due to the
45 reason that it is protected by lignin and hemicellulose whereas in pretreated samples the lignin and
46
47 hemicellulose removal improved its accessibility (Taherzadeh and Karimi, 2008). The lignin
48
49 reduction during pretreatment was directly correlated with the cellulose degradation as shown in
50
51 Fig 6. In NaOH pretreated manure fibers the ester bonds between lignin and carbohydrates is
52
53 damaged because of saponification reactions during pretreatment. Due to these saponification
54 reactions the cellulose fraction is exposed and can be attacked by anaerobic bacteria. While the
55
56 hydrogen bonds of cellulose is resistant to anaerobic digestion the NaOH pretreatment breaks these
57
58 bonds which may cause significant higher cellulose conversion compared to untreated samples
59
60 (He et al., 2008).
61
62 13
63
64
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 1
2
3
4 Hemicellulose is an amorphous polymer and can be easily hydrolyzed compared to cellulose.
5
6 Table 2 shows that hemicellulose is affected more by pretreatment compared to cellulose and
7
8 lignin resulting in a higher hemicellulose conversion compared to cellulose conversion during the
9
10 AD process. The intermolecular hydrogen bonds between hemicellulose monomers was further
11
12 destroyed during NaOH pretreatment. Besides this β-glycosidic linkages between hemicellulose
13
14
sugar units was further broken during pretreatment, which may have increased its degradation
15 during anaerobic digestion process (Sun et al., 2000). Overall the NaOH assisted thermal
16
17 pretreatment lead to higher degradation during anaerobic digestion compared to control, physical,
18
19 chemical and thermal pretreated samples. The statistical analysis of the effect of pretreatment on
20
21 cellulose, hemicellulose and volatile solids degradation has been presented in Table 4. Again, upon
22
23 analyzing the table, we can state that all pretreatments had a significant effect on degradation of
24
25
cellulose, hemicellulose and volatile solids (P < 0.005).
26
27 4. Conclusion
28
29
30 In this study different pretreatments were used for the anaerobic digestion of digested manure
31 fibers. It was observed that thermal pretreated manure fibers with NaOH added showed higher
32
33 degradation and methane yield compared to other pretreatments. The NaOH pretreatment of
34
35 manure fibers separated out after primary anaerobic digestion led to an increase in the methane
36
37 yield during secondary AD of 127% along with an increase in the VS reduction of 42.3%.
38
39 Moreover, 76.6% of the cellulose and 84.8% of the hemicellulose was converted to methane.
40
41 Acknowledgements
42
43
44 This work was supported by a grant from WSU CAHNRS Appendix A program.
45
46
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Tsapekos, P., Kougias, P. G., Frison, A., Raga, R., Angelidaki, I. 2016. Improving methane
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32 Xiao, B., Sun, X., Sun, R., 2001. Chemical, structural, and thermal characterizations of alkali-
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38 Zhang, C., Li, J., Liu, C., Liu, X., Wang, J., Li, S. Zhang, L. 2013. Alkaline pretreatment for
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40 enhancement of biogas production from banana stem and swine manure by anaerobic codigestion.
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44 Zhao, X., Luo, K., Zhang, Y., Zheng, Z., Cai, Y., Wen, B, Wang, X. 2018. Improving the methane
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46 yield of maize straw: focus on the effects of pretreatment with fungi and their secreted enzymes
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Table.1 Pretreatment Conditions tested
16
17 Pretreatment NaOH (% w/w) Temperature HRT (min) TS (%)
18
19 Type (oC)
20
21 Control 0 25 0 10
22
23 Milling 0 25 3 10
24
Chemical 2 25 30 10
25
26 Thermal 0 180 30 10
27
28 Thermo- 1 180 30 10
29
30 Chemical-1
31
32 Thermo- 2 180 30 10
33
34 Chemical-2
35 Thermo- 3 180 30 10
36
37 Chemical-3
38
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Table.2 Compositional Analysis of Feed and Effluent
16
17 Pretreatment Sample Type Cellulose Hemicellulose Lignin
18 Condition
19
20 Control Feed 37.1±0.5 26.1±0.4 36.7±0.9
21 Effluent 13.8±0.2 9.08±0.1 42.1±0.7
22
23 Milling Feed 36.8±0.3 25.8±0.7 36.3±0.4
24
Effluent 12.5±0.7 5.7±0.2 39.6±1.2
25
26
27 Chemical Feed 35.4±1.1 24.9±0.6 36.2±0.4
28 (2%NaOH)
29 Effluent 12.1±0.9 5.4±0.3 38.9±0.6
30
31
32 Thermal Feed 35.1±0.8 23.3±1 36.2±0.9
33 Effluent 12.9±0.3 5.1±0.2 40.8±0.3
34
35 Thermochemical Feed 33.7±0.6 21.1±1.6 34.6±1.4
36
37 (1%NaOH)
38 Effluent 7.2±0.4 3.7±0.2 36.3±0.7
39 Thermochemical Feed 28.4±0.7 18.7±0.9 33.4±0.8
40 (2%NaOH)
41
42
Effluent 6.7±0.2 3.1±0.1 36.2±0.4
43 Thermochemical Feed 27.9±0.7 18.5±1.3 33.2±1.1
44
45 (3%NaOH)
46 Effluent 6.2±0.2 2.6±0.1 34.2±1.3
47
48
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16
17 Table.3 ANOVA Table for Effect of Pretreatment Condition on methane Yield
18
19
20 SS DF MS P Value
21
22
Pretreatment 410595 6 68433 P < 0.05
23 Condition
24
25 Days 235588 57 4133 P < 0.05
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
41
42
43
44
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9
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16
17 Table.4 ANOVA Table for Effect of Pretreatment Condition on cellulose, hemicellulose and
18
19 volatile solids degradation
20
21
SS DF MS P Value
22
23 Pretreatment 2148 6 358 P < 0.05
24
25 Condition
26
27 Degradation 7831 2 3916 P < 0.05
28
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17 Fig 1 Reduction of cellulose, hemicellulose and lignin as the result of different pretreatments
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21 Fig 2 Methane yield from the different semi-continuous anaerobic bioreactors
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23 Fig 3.Total biogas, total methane and methane yield (ml/gVS) from all the samples
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Credit Author Statement

Author Statement
Muhammad Usman Khan: Conceptualization, Formal analysis, Investigation, Data Curation,
Writing - Original Draft
Birgitte K. Ahring Conceptualization, Investigation, Supervision, Reviewing and Editing
Declaration of Interest Statement

To whom it may concern

The Authors hereby declare that the work presented herein is original and is not published anywhere. They
have no conflict of interest.

Sincerely,

Birgitte K. Ahring, Ph.D.

Battelle Distinguished Professor

Bioproducts, Sciences and Engineering Laboratory, BSEL

2710 Crimson Way, Richland, WA 99354-1671

509.372.7625 | www.tricities.wsu.edu