Advanced Chemical Design for Efficient Lignin Bioconversion - ACS

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Advanced Chemical Design for Efficient Lignin Bioconversion Shangxian Xie, Qining Sun, Yunqiao Pu, Furong Lin, Su Sun, Xin Wang, Arthur Jonas Ragauskas, and Joshua S. Yuan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02401 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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ACS Sustainable Chemistry & Engineering

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Advanced Chemical Design for Efficient Lignin Bioconversion

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Shangxian Xieabc, Qining Sund, Yunqiao Pudf, Furong Linabc, Su Sunabc, Xin Wangabc, Arthur

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Ragauskasdef, and Joshua S. Yuan*abc

4

a

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College Station, TX 77843, USA

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b

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77843, USA

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c

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77843, USA

Texas A&M Agrilife Synthetic and Systems Biology Innovation Hub, Texas A&M University,

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX

Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX

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d

11

TN 37996, USA

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e

13

USA

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f

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* For correspondence: [email protected], Phone: 979 845 3016, Complete Address: 2123 TAMU,

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College Station, TX 77843

Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville,

Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville, TN 37996,

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

17

1 ACS Paragon Plus Environment


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Abstract

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Lignin depolymerization mainly involves redox reactions relying on the effective electron

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transfer. Even though electron mediators were previously used for delignification of paper pulp,

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no study has established a bioprocess to fragment and solubilize the lignin with an effective

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laccase-mediator system, in particular, for subsequent microbial bioconversion. Efficient lignin

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depolymerization was achieved by screening proper electron mediators with laccase to attain

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nearly 6 fold increase of kraft lignin solubility comparing to the control kraft lignin without

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laccase treatment. Chemical analysis suggested the release of low molecularweight fraction of

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kraft lignin into solution phase. Moreover, NMR analysis revealed that an efficient enzyme-

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mediator system can promote the lignin degradation. More importantly, the fundamental

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mechanisms guided the development of an efficient lignin bioconversion process, where

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solubilized lignin from laccase-HBT treatment served as a superior substrate for bioconversion

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by Rhodococcus opacus PD630. The cell growth was increased by 106 fold, and the lipid titer

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reached 1.02 g/L. Overall, the study has manifested that an efficient enzyme-mediator-microbial

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system can be exploited to establish a bioprocess to solubilize lignin, cleave lignin linkages,

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modify the structure, and produce substrates amenable to bioconversion.

34 35 36

Keywords

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Laccase-Mediator-Microbial, Lignin, Valorization, Bioconversion, Rhodococcus

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Introduction Lignin valorization represents one of the most significant challenges for the sustainability

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and profitability of lignocellulosic biorefinery.1-4

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biopolymer in nature and a major waste stream from a lignocellulosic biorefinery and from the

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pulping and paper industry The utilization of the lignin-containing biorefinery waste for valuable

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products will improve CO2 mitigation, net energy gain, waste management, and cost-

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effectiveness of biofuels.2 The fundamental challenge for lignin depolymerization lies in the fact

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that lignin is a complex substituted polyphenol derived from hydroxycinnamyl monomers with

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various degrees of methoxylation forming a racemic, cross-linked, and highly heterogeneous

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aromatic macromolecule.1-2,

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among different plants, which make the design of effective lignin depolymerization more

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challenging. Even though many chemical and thermochemical processes have been developed

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for lignin depolymerization, most of these processes involved high temperature, high pressure,

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high energy cost and generating many inhibitors for microbial growth.

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depolymerization might have its advantage due to the mild condition and potentially fewer

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inhibitors for microbes. Unlike cellulose degraded primarily by the hydrolysis process,

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enzymatic lignin depolymerization heavily depends on redox reactions relying on the electron

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transfer from a proper donor to target substrate. Enzymatic lignin depolymerization is thus often

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mediated by various small molecule oxidants produced by enzymes, such as laccase and

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manganese peroxidase (MnP), which can react directly with lignin to generate radical sites

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within the substrate and initiate bond scission reactions. To that extent, the dependence on

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efficient electron transfer applies to all major biochemical lignin depolymerization systems like

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laccase, manganese peroxidase (MnP), and Fenton reactions.1

5

Lignin is the second most abundant

Lignin content and composition may also vary significantly


6-8

The enzymatic lignin


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We have recently demonstrated that laccase and R. opacus PD630 cell can synergize effective

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lignin degradation and conversion.9 Despite the progresses, enzymatic lignin depolymerization

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still rendered very low yield of fragmented and soluble lignin, partially due to the limitations on

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efficient electron transfer. In case of laccase, it can self-generate radicals by releasing the

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phenolic compounds from lignin to create a sustainable redox environment to achieve lignin

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depolymerization and modification. However, the phenolic radicals released during lignin

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oxidation by laccase were very limited and often insufficient to mediate the electron transfer for

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the efficient cleavage of different chemical bonds. The fundamental scientific challenge for

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effective enzymatic lignin depolymerization thus is to improve electron transfer during the redox

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reaction.

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Electron mediators are a class of chemicals having redox moieties that can facilitate the

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transfer of the electron or redox potential from donor to acceptor during a reaction. Traditionally,

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various laccase-mediator systems have been exploited to improve lignin removal in paper

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pulping, to degrade hazardous aromatics for environmental remediation, and to modify

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lignocellulosics in biorefinery pretreatment.10-13 Despite the progresses, the application of a

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laccase-mediator system in lignin conversion and the understanding of chemical mechanisms for

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recalcitrant lignin depolymerization by electron mediators are both still very limited. First, the

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concept of using an enzyme-mediator system to solubilize purified lignin toward bioconversion

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is yet to be proven. No study has established a process for lignin solubilization and conversion

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using an efficient laccase-mediator system. Neither is it known if development of such a process

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is attainable, as different electron mediators have various impacts on enzymatic oxidation of

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lignin, making the outcome of a biological-chemical catalytic system unpredictable. Second, the

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molecular and chemical mechanisms for electron mediator-facilitated lignin depolymerization


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are yet to be revealed. Previous studies mostly exploited model compounds to illustrate how an

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electron mediator could facilitate the cleavage of certain chemical bonds in the model

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compounds.14-15 Some studies also investigated the effects of laccase-mediator system on the

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lignin-carbohydrate complex during the delignification of paper pulp.16 However, very few

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studies have comprehensively revealed what chemical linkages the laccase-mediator system can

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efficiently degrade in purified lignin, which has hindered the application of such a system in

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lignin conversion and processing. Third, no research has established how the fragmented lignin

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out of laccase-mediator treatment can be integrated with microbial fermentation for

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bioconversion. Neither do we know if the solubilized lignin derived from laccase-mediator

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treatment can be compatible for microbial fermentation.

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In this study, we have addressed each of these three challenges both to establish an effective

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lignin conversion platform with an effective enzyme-mediator-microbial system and to elucidate

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the fundamental mechanisms for the efficient lignin degradation by such a system. The outcome

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reveals chemical mechanisms with broad scientific impacts on biomass utilization and delivers

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an effective bioprocess to achieve record level of bioproduct yield from lignin bioconversion.

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Results and Discussions

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Electron mediators could promote enzymatic fractionation and solubilization of kraft

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lignin in a mediator-specific way

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An efficient laccase-mediator system was first designed to solubilize >35% of insoluble kraft

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lignin. Kraft lignin is among the most recalcitrant and insoluble lignin. In general, laccase itself

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could only carry out oxidative attack on the phenol structure, which accounts for only a small

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portion of lignin.17 To achieve effective lignin depolymerization, it is necessary to establish an

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efficient laccase-mediator system, where the electron mediator can facilitate the redox transfer



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during the oxidation by laccase and the subsequent non-enzymatic attack on non-phenolic lignin

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(Figure 1).17-19 However, different mediators may have various impacts on the laccase-based

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lignin depolymerization reaction and could promote either polymerization or depolymerization

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reactions. The impact of electron mediators on lignin depolymerization is therefore subject to

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debate and the outcome of a given enzyme-mediator system is unpredictable. In order to address

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these challenges, we first screened four different types of mediators in combination with a well-

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studied commercial laccase from Trametes versicolor. In order to well integrate with downstream

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bioconversion by bacteria Rhodococcus opacus PD630, the pH for lignin depolymerization by

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laccase-mediator system was adjusted to 7.0. As shown in Figure 2A, the weight loss analysis

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revealed that most of the electron mediators promoted lignin depolymerization. As compared to

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the laccase treatment control, essentially all electron mediators can increase the solubilization of

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kraft lignin in water (Figure 2A, Table S1).

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However, the efficiency for lignin depolymerization varies substantially among different

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mediators (Figure 2). For example, the laccase-HBT (1-hydroxybenzotriazole) system could lead

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to the most efficient lignin depolymerization with more than 35% of the kraft lignin released into

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water. Only about 15%, 22% and 13% of lignin was solubilized, when ABTS (2,2'-azino-bis(3-

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ethylbenzothiazoline-6-sulphonic acid)), acetosyringone, and phenol were used as mediators,

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respectively. The data from weight loss analysis was further confirmed by Prussian Blue assay of

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total aromatic compounds solubilized (Figure 2B). As shown in Figure 2B, the laccase-HBT

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system led to the highest concentration of aromatic compounds in water. The GC/MS analysis

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was further carried out to analyze the soluble monomer compounds released after lignin

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depolymerization by different laccase-mediator systems (Figure 2C). The heatmap presented the

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cluster analysis of normalized relative quantity of the various monomer compounds in the


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solution phase. The cluster analysis indicated that the compounds being clustered in the same

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group may involve similar conversion pathway or mechanism. The GC/MS analysis revealed

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that laccase in combination with various mediators released different aromatic monomer

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compounds, indicating the selective targeting of different chemical linkages during lignin

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oxidation.

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Overall, the results highlighted two features for electron mediators to promote enzymatic

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lignin depolymerization through more efficient redox reactions toward enzymatic lignin

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depolymerization. First, an efficient laccase-mediator system could substantially promote lignin

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fragmentation and solubilization.

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selectivity for facilitating the lignin depolymerization reactions. We therefore focused on the

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most efficient mediator, HBT, to study the chemical mechanisms for electron mediators to

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promote lignin degradation and to investigate the feasibility of coupling laccase-mediator-based

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lignin solubilization with microbial bioconversion.

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Molecular weight of different lignin fractions after enzyme-mediator treatment suggested

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the release of low molecular weight fraction in solution phase

Second, various mediators have different efficiency and

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In an effort to reveal the mechanisms for laccase-mediator-based lignin depolymerization, gel

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permeation chromatography (GPC) was employed to analyze molecular weight distribution of

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the insoluble and soluble fraction of kraft lignin before and after the laccase-mediator treatment.

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There was a significant increase of molecular weights of insoluble lignin after the laccase and

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laccase-HBT treatment. Mw of lignin increased from 3122 to 5595 and 5337 g/mol for laccase

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and laccase-HBT treatment, respectively. Mn of lignin increased from 801 to 1678 and 1652

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g/mol for laccase and laccase-HBT treatment, respectively. (Figure 3A, 3B). GPC analysis also

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revealed the decrease of polydispersity index (PDI) in the insoluble fraction of the treated lignin



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(Figure 3C), which could be attributed to the behavior of Mn and Mw. PDI was obtained through

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dividing Mw by Mn, and any changes on those two parameters could result in the altered PDI.

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The decreased PDI suggested that the insoluble lignin after laccase and mediator treatment

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became more uniform in molecular weight distribution.

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On one side, the increase in average molecular weights and uniformity could result from

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repolymerization and re-arrangement of lignin structure by laccase.20 On the other side, the

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increased molecular weights could also be attributed to the better fragmentation of lower mass

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lignin and the solubilization of such released small molecular weight compounds during lignin

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depolymerization. Considering that the laccase and mediator treatments led to increased

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solubilization of kraft lignin (Figure 2), it is likely that the degradation of low molecular weight

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lignin at least contributed partially to the increased molecular weight for insoluble lignin. GPC

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analysis was carried to analyze the soluble fraction and further confirm the enhanced release of

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low molecular weight compounds by laccase-mediator systems. The soluble fractions of lignin

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under laccase and laccase-HBT treatments were compared with that of the untreated lignin. As

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shown in Figure S1 and Table S2, GPC analysis of the soluble fraction showed that the released

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soluble products had a number-averaged and weight-average molecular weights of 135 g/mol

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and 646 g/mol for laccase treated lignin, 192 g/mol and 370 g/mol for laccase-HBT treated

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lignin, respectively. The results further confirmed the release of low molecular weights products.

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Overall, GPC analysis confirmed that laccase-mediator systems could promote the lignin

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fragmentation and solubilization by releasing low molecular weight lignin fraction into the liquid

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phase. The

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degradation of abundant chemical groups contributed to the increased molecular weight and

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uniformity of insoluble lignin (Table 1).

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P-NMR (Nuclear Magnetic Resonance) analysis further verified that the


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Lignin degradation by laccase-mediator system as demonstrated by NMR To further investigate the fundamental mechanisms of lignin solubilization during laccase

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and laccase-HBT treatments, quantitative

P NMR technique was applied to monitor the

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changes of aliphatic, phenolic hydroxylic and carboxylic functional groups in kraft lignin. The

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to illustrate the effects of laccase-HBT system on lignin chemical bonds cleavage. As shown in

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Table 1, the kraft lignin used in the study was mainly composed of guaiacyl (G) and p-

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hydroxyphenyl (H) units, as revealed by the presence of G and H spectra signal and the absence

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of syringyl (S) spectra signal. The

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lead to much more significantly decrease of non-condensed structures including guaiacyl, p-

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hydroxyphenyl and aliphatic OH structure by 42.1%, 45.5%

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compared to those of untreated lignin. The decrease of these functional groups by laccase alone

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treatment was much less significant, where only 27.9% decrease of guaiacyl group was observed,

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and no significant changes of p-hydroxyphenyl structure and aliphatic OH structure were found

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(Table 1). The laccase-mediator system was reported preferentially to attack phenolic substrates.

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The reduction in phenolic lignin moieties has also been observed by Sealey and Ragauskas who

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characterized the natural of the residual lignin in a softwood kraft pulp before and after a laccase-

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HBT treatment.21 Similarly, Poppius-Levlin et al. reported a substantial decrease (by 42%) of

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phenolic groups for the residual lignin isolated from the laccase-HBT treated pulp.22 Moreover,

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the C5 substituted condensed phenolic structure (i.e., β-5, 4-O-5 and 5-5) in kraft lignin were

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significantly decreased after laccase-HBT treatment by ~37.2%. However, only 13.5% decrease

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of C5 condensed structure was observed in laccase alone (Table 1). Tamminent et al. also studied

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the reactivity of laccase/HBT towards phenolic condensed lignin structures demonstrating a

P NMR analysis of insoluble fraction of kraft lignin provided an accurate and quantitative way

31

P NMR revealed that laccase-HBT system treatment could


and 26.1%, respectively, as


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decrease in phenolic 5-5 condensed structures in a pine kraft pulp treated with laccase/HBT.

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Meanwhile, carboxylic group in the insoluble lignin after laccase-HBT treatment were found

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significantly decreased as compared to those in untreated lignin and laccase-treated lignin. These

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results strongly suggested that laccase-HBT could degrade lignin in a much higher efficiency

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than that of laccase alone treatment.

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The electron mediator-facilitated lignin degradation was further investigated by the 2D-

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heteronuclear single quantum coherence (HSQC) NMR. The major inter-units observed in

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untreated kraft lignin were the β-O-4 (A), β-5/α-O-4 (B), and β-β (C) (Figure S2). The HSQC

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spectra showed a similar pattern of major interunit linkages in insoluable lignin fractions after

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laccase and laccase-HBT treatment as compared to the untreated control, suggesting a similar

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lignin core structures remained. The assessment of cross-peak intensity qualitatively indicated a

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decrease in intensity of both aliphatic region (β-aryl ether linkages, phenylcoumaran as well as

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resinol units) and aromatic region (the major guaiacyl unit) for both laccase and laccase-HBT

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treated lignins (Figure S2). The relative abundance of main interunit linkages in the untreated

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and laccase-HBT treated kraft lignin samples was estimated by semi-quantitative analysis of

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HSQC spectra following reported procedures.23-24. The HSQC analysis showed a slight decrease

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in the relative abundance of β -O-4 linkage in the laccase and laccase-HBT treated lignin when

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compared to that in the untreated kraft lignin. Laccase-HBT system has been reported to be able

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to degrade non-phenolic β-O-4 lignin model structures. The result efficient lignin

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depolymerization by laccase-HBT treatment might be related to the cleavage of β-O-4 linkage.25

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The solubilized lignin by laccase-HBT system significantly promoted cell growth and lipid

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yield during bioconversion


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We further investigated if the solubilized lignin from laccase-mediator system can be used for

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bioconversion.

Although

recent

technology

breakthroughs

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chemical/physical methods to pre-process lignin for efficient fractionation,26-28 the combination

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of these methods with bioconversion leads to limited increase of bioproduct yield from lignin as

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compared to that from model compounds.29 Considering the potentially less toxic compounds

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produced by enzymatic lignin fragmentation as compared to chemical fragmentation, we

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investigated the compatibility of this laccase-mediator system with microbial bioconversion. The

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soluble fraction of laccase-HBT depolymerized kraft lignin was used as fermentation substrate

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for an oleaginous bacteria R. opacus PD630. The results highlighted that the lignin

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depolymerization by the laccase-mediator treatment could significantly promote lignin

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consumption and enable a record level of bioproduct yield on lignin. When the solubilized lignin

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was used as the sole carbon source for R. opacus PD630 fermentation, the cell growth was

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increased by 106 folds as compared to the cells grown on untreated kraft lignin based on colony-

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forming unit (CFU) (Figure 4A). The cell dry weight reached to 2.79 g/L, representing the

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highest yield for microbial fermentation of lignin. The lipid yield achieved 1.02 g/L, which was

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10 time higher than the cells grown on untreated lignin. The GC/MS analysis of the aromatic

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monomer compounds in the medium before and after fermentation indicated the consumption of

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several major compounds including cinnamic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic

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acid, phenol, vanillin, and others. The capacity of R. opacus PD630 to use the lignin derived

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aromatic compounds was furthered conformed by cultivating the bacteria in the model

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compounds, including vanillin, phenol, cinnamic acid and 4-hydroxybenzoic acid, as sole carbon

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source (Figure S3). In addition, several new aromatic compounds like 1,3-diacetylbenzene,

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octopamine and 1,4-cyclohexadiene were also found in the media after the fermentation. The


have

enabled

several


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results indicated that R. opacus PD630 not only utilized the aromatic monomers, but also

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degraded the aromatic oligomers to release monomers. Overall, the significant increases in lipid

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yield and cell growth indicated that the enzyme-mediator system for lignin fragmentation was

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very compatible with a variety of bioconversion platforms to process highly recalcitrant and

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insoluble lignin like kraft lignin.

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The chemical mechanisms for efficient lignin depolymerization catalyzed by an enzyme-

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mediator system

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The biological and chemical analyses suggested several aspects of the fundamental

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mechanisms for electron mediators to facilitate enzymatic lignin depolymerization. First, weight

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loss and Prussian Blue assay of lignin solubilization suggested that the enhanced electron

255

transfer could promote enzymatic depolymerization of insoluble and recalcitrant kraft lignin. The

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various electron mediators showed different selectivity and efficiency to interact with the same

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laccase for lignin depolymerization. Second, the GPC analysis of different fractions of kraft

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lignin after treatment suggested the more efficient release of low molecular weight fraction of

259

lignin by laccase-mediator system. Third, the NMR analyses revealed that the electron mediators

260

like HBT can facilitate the electron transfer to promote the cleavage of a broad range of chemical

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linkages within lignin, which further resulted in the enzymatic degradation of different abundant

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functional groups.

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The enhanced redox reactions for chemical bond cleavage were the basis for more efficient

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enzymatic lignin depolymerization. Even though previous studies revealed the improved

265

cleavage of certain chemical bonds by laccase-mediator system, this study for the first time

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revealed the efficient synergy between laccase-mediator-microbial system for lignin

267

bioconversion. The enhanced lignin fragmentation and solubilization was likely to be due to both


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the more rapid electron transfer and the more efficient penetration of redox active moieties into

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lignin macromolecules. The fundamental mechanisms can be further studied, yet they already

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can be translated into broad applications including lignin conversion, selectively modification of

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lignin structure, and preparation of lignin for the synthesis of various carbon materials.

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Electron-mediator system can have broad application in lignin processing

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The scientific discovery has enabled an effective platform for lignin processing. First, the

274

study established a lignin solubilization process by an efficient laccase-mediator system. Despite

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the abundance and importance, lignin utilization is hindered by the lack of efficient

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fragmentation and/or depolymerization methods to produce oligomers and monomers for

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bioconversion or chemical synthesis.1-2 The design of an efficient laccase-mediator system has

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enabled the solubilization of 35% of highly recalcitrant kraft lignin, resulting in a water soluble

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fraction as well as a more condensed and uniform insoluble fraction of lignin. Second and more

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importantly, we have demonstrated the compatibility of the soluble lignin fraction with

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bioconversion using R. opacus. Bioconversion of lignin into various bioproducts has emerged as

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a powerful platform.5, 30 However, the bioproduct yields from untreated lignin are usually very

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low as compared to those on model compounds.30 Even though several chemical processes were

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developed for lignin depolymerization, the combination of these processes with microbial

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bioconversion still could not achieve bioproduct titer higher than 1 g/L, assumingly due to the

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toxic compounds produced under extreme chemical conditions (Table S3).26,

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depolymerization of lignin serves as a viable alternative to produce fragmented products that are

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less toxic and more amenable to bioconversion. Another significant advantage for the enzymatic

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process is the relevant mild conditions, which will significant reduce the capital investment for

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the bioprocess stream in an integrated biorefinery setting. Despite the potential, enzymes like


28-29

Enzymatic


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laccase can only lead to very limited lignin depolymerization. The enhanced electron transfer and

292

resultant cleavage of abundant chemical linkages by proper mediators significantly improved

293

enzymatic lignin depolymerization and subsequent release of soluble monomers and oligomers

294

which could be served as carbon source for microbial lipid conversion. The significant improved

295

lipid yield and cell growth on laccase-HBT treated lignin indicated that the new enzyme-

296

mediator platform could be broadly applied to bioconversion of lignin into different bioproducts.

297

Conclusion

298

The utilization of lignin-containing waste stream for fungible bioproducts represents a critical challenge

299

for integrated biorefinery and paper pulping industry. Inevitably, an effective biorefinery stream derived

300

from the traditional biorefinery waste will increase the cost-effectiveness, carbon and energy efficiency to

301

enable the lignocellulosic biofuel. In this study, we achieved 35% of the lignin to be depolymerized and

302

solubilized into water, which could be used by oleaginous bacteria R. opacus PD630 as sole carbon

303

source and conversed into lipid with a product yield of 1.02 g/L. The lignin characterization including

304

31

305

mediator system. These findings advanced the understanding of lignin depolymerization and developing

306

of the lignin valorization by microbial conversion.

307

Materials and Methods

308

Lignin depolymerization by laccase-mediator

P-NMR and GPC further confirmed the more efficient and broader chemical binds cleavage by laccase-

309

The alkali lignin (catalog number 370959) and laccase from Trametes versicolor (catalog

310

number 51639) used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

311

For lignin depolymerization experiment, 6% (w/v) of lignin was loaded in 35 mM phosphate

312

buffer (pH 7.0) and mixed with laccase with a loading of 150 U per gram of lignin and 1mM of

313

the proper electron mediator as discussed in the manuscript. The mixture was incubated for 24


Page 15 of 32

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314

hours at 50 ℃ with a shaking speed of 180 rpm. The solubilized lignin after laccase-mediator

315

depolymerization was collected by centrifuging at 10,000 g for 15 min. The collected solubilized

316

lignin was used for further Gas Chromatography–Mass Spectrometry (GC-MS) analysis,

317

Prussian Blue assay and lignin fermentation. The insoluble lignin from the pellet after centrifuge

318

was washed twice with double distilled water and dried at 65 ℃ to reach constant weight. The

319

weight loss of the insoluble lignin was calculated to determine the lignin solubilization efficiency.

320

The dried insoluble lignin was also characterized by GPC and 31P-NMR.

321

GC-MS analysis of the solubilized lignin

322

The monomer composition of the solubilized lignin was analyzed by GC-MS with the

323

method modified from previous study.31 3 mL of solubilized lignin was extracted with 9 mL of

324

ethyl acetate by vigorously shaking for 2 hours. The organic layer was collected and dewatered

325

using anhydrous sodium sulfate. 1 mL of the dewatered solution was collected after centrifuging

326

at 4,000 g for 10 min and dried under nitrogen gas stream. The dried residue was mixed with 100

327

µL dioxane, 10 µL pyridine and 50 µL trimethyl silyl. The mixture was incubated at 60 ℃ for 15

328

min with periodic shaking to completely dissolve the residue. 1 µL of the silylated compounds

329

were injected into SHIMADZU-QP2010 SE GC-MS equipped with a ZB-5MSi column

330

(thickness 0.25 µm; length 30m; diameter 0.25 mm). The column temperature program was set

331

as: 50 ℃ hold for 5 min; 50-300 ℃ (10 ℃/min, hold time: 5 min). The low molecular compounds

332

were identified by searching against the NIST library available with the instrument.

333

Lignin concentration analysis by Prussian Blue assay

334

The concentration of total solubilized lignin in the solution after laccase-mediator treatment

335

or bacterial fermentation was estimated by Prussian Blue assay.32-33 The solubilized lignin was

336

first diluted to an optimal concentration using ddH2O to a final volume of 1.5mL and mixed



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

337

with 100 µL of 8 mM K3Fe(CN)6 and 100 µL 0.1 M FeCl3. The samples were mixed

338

immediately and reacted for exact 5 min. The samples were transferred to 1cm cuvette to record

339

the absorbance reading at 700 nm by spectrophotometer. Standard curve was established with the

340

same reagents and known concentration of catechol. All experiments were carried out in

341

triplicate.

342

Lignin characterization by GPC

Page 16 of 32

343

The molecular weight of the soluble and insoluble fraction of lignin before and after

344

treatment was analyzed by GPC. The lignin (20 mg) was first acetylated by incubating with

345

acetic anhydride/ pyridine (1:1 v/v, 1.0 mL) for 24 h in a sealed flask under an inert atmosphere

346

at room temperature according to the literature.34 After 24 h, the solution was diluted with 20 mL

347

of ethanol and stirred for an additional 30 min, after which the solvents were removed with a

348

rotary evaporator followed by drying in a vacuum oven at 40 °C. Prior to GPC analysis the

349

acetylated lignin sample was dissolved in tetrahydrofuran (1.0 mg/mL), filtered through a 0.45

350

µm filter, and placed in a 2 mL auto-sampler vial. The molecular weight distributions of the

351

acetylated lignin samples were then analyzed on an Agilent GPC SECurity 1200 system

352

equipped with four Waters Styragel columns (HR1, HR2, HR4, HR6), an Agilent refractive

353

index (RI) detector, and an Agilent UV detector (270 nm). Tetrahydrofuran was used as the

354

mobile phase in GPC analysis and the flow rate was 1.0 mL/min. A standard polystyrene sample

355

was used for calibration. The number-average molecular weight (Mn) and weight-average

356

molecular weight (Mw) were determined by GPC. Average data of Mw, Mn and polydispersity

357

index (PDI) were presented based on two different tests on each lignin sample.

358

Lignin characterization by NMR


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359

The more detailed changes in chemical linkages of lignin by different treatment were

360

analyzed by NMR. HSQC-NMR were acquired at a 500 MHz Varian VNMRS using 50 mg

361

lignin dissolved in 500 uL dimethyl sulfoxide-d6 (DMSO-d6) employing a standard sequence

362

“Gradient HSQCAD” with 0.15s acquisition time, a 1.0 s pulse delay, a 1JC-H of 146 Hz, 48 scan

363

and acquisition of 1024 data point (for 1H) and 256 increments (for

364

peak was used for chemical shift calibration. For quantitative

365

31

13

C). The central solvent

P NMR analysis, 20 mg lignin was dissolved in 500 µL pyridine/CDCl3

366

(1.6/1.0 v/v) solvent containing chromium acetylacetonate (relaxation agent) and endo-N-

367

hydroxy-5-norbornene-2,3-dicarboximide (NHND, internal standard), then derivatized with 2-

368

chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane. The spectrum was acquired using an

369

inverse-gated decoupling pulse sequence (Waltz-16), a 90° pulse, and a 25-s pulse delay. 128

370

scans were accumulated for each sample. NMR data were processed using the software of

371

TopSpin 2.1 (Bruker BioSpin) and MestreNova (Mestre Labs) packages.35

372

Strain and culture medium

373

R. opacus PD630 (DSMZ 44193) was purchased from the DSMZ-German Collection of

374

Microorganisms and Cell Cultures at the Leibniz Institute. The culture medium was modified

375

from previous publication36 which contained (per liter): 1.4 g (NH4)2SO4, 1.0 g MgSO4·7H2O,

376

0.015 g CaCl2·2H2O, 35.2 mL sterile 1 M pH 7.0 phosphate buffer (113 g/L K2HPO4 and 47 g/L

377

KH2PO4), 1.0 mL sterile trace element solution (0.5 g/L FeSO4·7 H2O, 0.4 g/L ZnSO4·7 H2O,

378

0.02 g/L MnSO4·H2O, 0.015 g/L H3BO3, 0.01 g/L NiCl2·6H2O, 0.25 g/L EDTA, 0.05 g/L

379

CoCl2·6H2O and 0.005 g/L CuCl2·2H2O), 1.0 mL sterile stock A solution (2.0 g/L

380

NaMoO2·2H2O and 5.0 g/L FeNa·EDTA) and certain carbon source.

381

Lignin fermentation



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Page 18 of 32

382

R. opacus PD630 was pre-cultured in aforementioned minimum medium with 0.5% glucose

383

as carbon source at 28 °C to OD600 1.5. The cells were harvested by centrifugation and washed

384

twice with equal volume of the culture medium without carbon source. 5 ml of the resuspended

385

cells were inoculated to 50 ml of aforementioned minimum medium with 1% of lignin as sole

386

carbon source. The fermentation was set up at 28 °C with shaking speed of 200 rpm, and the

387

samples were harvested periodically for bacterial growth assay and lipid extraction.

388

Cell growth and total lipid yield measurement

389

Considering that the cells and untreated lignin (with significant portion of insoluble contents)

390

cannot be readily separated, it is difficult to measure either the OD600 or dry cell weight of R.

391

opacus PD630 grown on untreated insoluble kraft lignin. Therefore, the growth of R. opacus

392

PD630 on both untreated kraft lignin and treated soluble lignin were estimated according to the

393

CFU.

394

The total lipid of R. opacus PD630 after lignin fermentation was extracted in the form of

395

fatty acid methyl ester (FAME) according to the method described in our previous study.37-38 The

396

bacteria cells were harvested by centrifugation after lignin fermentation and incubated with 20

397

mL methanol at 65 °C for 30 min. 1 mL of 10 M KOH was added and incubated at 65 °C for 2 h.

398

After the incubation, the sample was moved out from water bath and cooled down to room

399

temperature. 1 mL of sulfuric acid was then added drop by drop and incubated at 65 °C for

400

another 2 h. 8 mL of hexane was added and incubated with periodically shaking for 5 min to

401

extract the lipid. The hexane layer was collected to pre-weighted glass tube after centrifugation

402

and this hexane extraction step was repeated once. The hexane was evaporated under nitrogen

403

gas stream and the lipid yield was calculated according to tube weight change. The CFU analysis

404

revealed a higher fold of cell growth increase as compared to the lipid yield, which could be due


Page 19 of 32

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405

to the larger variation in measuring lipid yield using weight-based methods at the low lipid titer

406

on untreated lignin.

407



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Page 20 of 32

408

Acknowledgement

409

The work was supported by the U.S. DOE (Department of Energy) EERE (Energy Efficiency

410

and Renewable Energy) BETO (Bioenergy Technology Office) (grant No. DE-EE0006112) to

411

JSY and AR. The research was also supported by Texas A&M Agrilife Research's biofuel

412

initiative to JSY.

413


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414

Supporting Information

415 416

Table S1. The estimated amount of total solubilized aromatic compounds after laccase treatment with different.

417 418

Table S2. The number-average molecular weights (Mn) and weight-average molecular weights (Mw) of soluble fraction of lignin after enzyme and enzyme-mediator treatments.

419 420

Table S3. Comparison of the lignin bioconversion efficiency with different lignin depolymerization methods.

421 422

Figure S1. GPC chromatogram of soluble fractions of control, laccase-treated and laccase-HBT treated lignin.

423 424 425

Figure S2. HSQC-NMR spectra (aliphatic regions) of the insoluble fractions of the untreated kraft lignin (Control), laccase treated kraft lignin (Laccase) and laccase-HBT treated kraft lignin (Lac-HBT).

426 427

Figure S3. The growth curve of R. opacus PD630 on 0.1% (w/v) aromatic model compounds as sole carbon source.

428



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Reference

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475

1. Xie, S.; Syrenne, R.; Sun, S.; Yuan, J. S., Exploration of Natural Biomass Utilization Systems (NBUS) for advanced biofuel-from systems biology to synthetic design. Curr. Opin. Biotechnol. 2014, 27, 195-203. 2. Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.; Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E., Lignin valorization: improving lignin processing in the biorefinery. Science 2014, 344 (6185). 3. Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T., The path forward for biofuels and biomaterials. Science 2006, 311 (5760), 484-489. 4. Xie, S.; Ragauskas, A. J.; Yuan, J. S., Lignin conversion: opportunities and challenges for the integrated biorefinery. Ind. Biotechnol. 2016, 12 (3), 161-167. 5. Xie, S.; Qin, X.; Cheng, Y.; Laskar, D.; Qiao, W.; Sun, S.; Reyes, L. H.; Wang, X.; Dai, S. Y.; Sattler, S. E.; Kao, K.; Yang, B.; Zhang, X.; Yuan, J. S., Simultaneous conversion of all cell wall components by an oleaginous fungus without chemi-physical pretreatment. Green Chem. 2015, 17 (3), 1657-1667. 6. Upton, B. M.; Kasko, A. M., Strategies for the conversion of lignin to high-value polymeric materials: review and perspective. Chem. Rev. 2015, 116 (4), 2275-2306. 7. Pandey, M. P.; Kim, C. S., Lignin depolymerization and conversion: a review of thermochemical methods. Chem. Eng. Technol. 2011, 34 (1), 29-41. 8. Wang, H.; Tucker, M.; Ji, Y., Recent development in chemical depolymerization of lignin: a review. J. Appl. Chem. 2013, 9. 9. Zhao, C.; Xie, S.; Pu, Y.; Zhang, R.; Huang, F.; Ragauskas, A.; Yuan, J. S., Synergistic enzymatic and microbial conversion of lignin for lipid. Green Chem. 2016,18, 1306-1312. 10. Babot, E. D.; Rico, A.; Rencoret, J.; Kalum, L.; Lund, H.; Romero, J.; del Río, J. C.; Martínez, Á. T.; Gutiérrez, A., Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator. Bioresour. Technol. 2011, 102 (12), 67176722. 11. Murugesan, K.; Chang, Y.-Y.; Kim, Y.-M.; Jeon, J.-R.; Kim, E.-J.; Chang, Y.-S., Enhanced transformation of triclosan by laccase in the presence of redox mediators. Water Res. 2010, 44 (1), 298308. 12. Rico, A.; Rencoret, J.; del Rio, J.; Martinez, A.; Gutierrez, A., Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels 2014, 7 (1), 6. 13. Rodríguez Couto, S.; Toca Herrera, J. L., Industrial and biotechnological applications of laccases: A review. Biotechnol. Adv. 2006, 24 (5), 500-513. 14. Li, K.; Xu, F.; Eriksson, K.-E. L., Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl. Environ. Microbiol. 1999, 65 (6), 2654-2660. 15. Shiraishi, T.; Sannami, Y.; Kamitakahara, H.; Takano, T., Comparison of a series of laccase mediators in the electro-oxidation reactions of non-phenolic lignin model compounds. Electrochim. Acta 2013, 106, 440-446. 16. Du, X.; Li, J.; Gellerstedt, G.; Rencoret, J.; Del Río, J. C.; Martínez, A. T.; Gutiérrez, A., Understanding pulp delignification by laccase–mediator systems through isolation and characterization of lignin–carbohydrate complexes. Biomacromolecules 2013, 14 (9), 3073-3080. 17. Bourbonnais, R.; Paice, M. G., Oxidation of non-phenolic substrates: An expanded role for laccase in lignin biodegradation. FEBS Lett. 1990, 267 (1), 99-102. 18. Eggert, C.; Temp, U.; Dean, J. F. D.; Eriksson, K.-E. L., A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett. 1996, 391 (1–2), 144-148. 22 ACS Paragon Plus Environment

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

476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523


19. Bourbonnais, R.; Leech, D.; Paice, M. G., Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. BBA-Gen. Subjects 1998, 1379 (3), 381-390. 20. Areskogh, D.; Li, J.; Gellerstedt, G.; Henriksson, G., Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Biomacromolecules 2010, 11 (4), 904-910. 21. Sealey, J.; Ragauskas, A. J., Residual lignin studies of laccase-delignified kraft pulps. Enzyme Microb. Technol. 1998, 23 (7), 422-426. 22. Poppius-Levlin, K.; Wang, W.; Tamminen, T.; Hortling, B.; Viikari, L.; Niku-Paavola, M.-L., Effects of laccase/HBT treatment on pulp and lignin structures. J. Pulp Paper Sci. 1999, 25 (3), 90-94. 23. Constant, S.; Wienk, H. L. J.; Frissen, A. E.; Peinder, P. d.; Boelens, R.; van Es, D. S.; Grisel, R. J. H.; Weckhuysen, B. M.; Huijgen, W. J. J.; Gosselink, R. J. A.; Bruijnincx, P. C. A., New insights into the structure and composition of technical lignins: a comparative characterisation study. Green Chem. 2016, 18, 2651-2665 24. Wen, J.-L.; Sun, S.-L.; Xue, B.-L.; Sun, R.-C., Recent advances in characterization of lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology. Materials 2013, 6 (1), 359-391. 25. Reiter, J.; Strittmatter, H.; Wiemann, L. O.; Schieder, D.; Sieber, V., Enzymatic cleavage of lignin [small beta]-O-4 aryl ether bonds via net internal hydrogen transfer. Green Chem. 2013, 15 (5), 13731381. 26. Rahimi, A.; Ulbrich, A.; Coon, J. J.; Stahl, S. S., Formic-acid-induced depolymerization of oxidized lignin to aromatics. Nature 2014, 515 (7526), 249-252. 27. Wei, Z.; Zeng, G.; Huang, F.; Kosa, M.; Huang, D.; Ragauskas, A. J., Bioconversion of oxygenpretreated Kraft lignin to microbial lipid with oleaginous Rhodococcus opacus DSM 1069. Green Chem. 2015, 17 (5), 2784-2789. 28. Linger, J. G.; Vardon, D. R.; Guarnieri, M. T.; Karp, E. M.; Hunsinger, G. B.; Franden, M. A.; Johnson, C. W.; Chupka, G.; Strathmann, T. J.; Pienkos, P. T.; Beckham, G. T., Lignin valorization through integrated biological funneling and chemical catalysis. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (33), 12013-8. 29. Vardon, D. R.; Franden, M. A.; Johnson, C. W.; Karp, E. M.; Guarnieri, M. T.; Linger, J. G.; Salm, M. J.; Strathmann, T. J.; Beckham, G. T., Adipic acid production from lignin. Energy Environ. Sci. 2015, 8 (2), 617-628. 30. Kosa, M.; Ragauskas, A. J., Lignin to lipid bioconversion by oleaginous Rhodococci. Green Chem. 2013, 15 (8), 2070-2074. 31. Raj, A.; Chandra, R.; Reddy, M. M. K.; Purohit, H.; Kapley, A., Biodegradation of kraft lignin by a newly isolated bacterial strain, Aneurinibacillus aneurinilyticus from the sludge of a pulp paper mill. World J.Microb. Biot. 2007, 23 (6), 793-799. 32. Budini, R.; Tonelli, D.; Girotti, S., Analysis of total phenols using the Prussian Blue method. J. Agric. Food Chem. 1980, 28 (6), 1236-1238. 33. Li, X.; Ximenes, E.; Kim, Y.; Slininger, M.; Meilan, R.; Ladisch, M.; Chapple, C., Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment. Biotechnol. Biofuels 2010, 3 (27), 1754-6834. 34. Trajano, H. L.; Engle, N. L.; Foston, M.; Ragauskas, A. J.; Tschaplinski, T. J.; Wyman, C. E., The fate of lignin during hydrothermal pretreatment. Biotechnol. Biofuels 2013, 6 (1), 110. 35. Pu, Y.; Cao, S.; Ragauskas, A. J., Application of quantitative 31P NMR in biomass lignin and biofuel precursors characterization. Energy Environ. Sci. 2011, 4 (9), 3154-3166. 36. Kurosawa, K.; Boccazzi, P.; de Almeida, N. M.; Sinskey, A. J., High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J. Biotechnol. 2010, 147 (3–4), 212-218. 37. Xie, S.; Sun, S.; Dai, S. Y.; S.Yuan, J., Efficient coagulation of microalgae in cultures with filamentous fungi. Algal Res. 2013, 2 (1), 28-33. 23 ACS Paragon Plus Environment


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

524 525 526 527 528 529 530 531 532 533 534 535

38. XIE, S.; YUAN, J., System and method of co-cultivating microalgae with fungus. Google Patents: 2012. 39. Kawai, S.; Nakagawa, M.; Ohashi, H., Aromatic ring cleavage of a non‐phenolic β‐O‐4 lignin model dimer by laccase of Trametes versicolor in the presence of 1‐hydroxybenzotriazole. FEBS Lett. 1999, 446 (2-3), 355-358. 40. Kawai, S.; Asukai, M.; Ohya, N.; Okita, K.; Ito, T.; Ohashi, H., Degradation of a non-phenolic β-O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the presence of 1-hydroxybenzotriazole. FEMS Microbiol. Lett. 1999, 170 (1), 51-57. 41. Srebotnik, E.; Hammel, K. E., Degradation of nonphenolic lignin by the laccase/1hydroxybenzotriazole system. J. Biotechnol. 2000, 81 (2), 179-188. 42. ten Have, R.; Teunissen, P. J., Oxidative mechanisms involved in lignin degradation by white-rot fungi. Chem. Rev. 2001, 101 (11), 3397-3414.

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Figure Legend Table 1. The abundance of functional groups in insoluble fraction of lignin before and after laccase and laccase-HBT treatment as revealed by 31P NMR analysis. Figure 1. The proposed lignin depolymerization mechanisms by a laccase-mediator system as summarized from previous studies.39-42 Figure 2. The solubilization of kraft lignin by different laccase-mediator systems. (A) The percentage of the solubilized lignin as calculated from the weight loss of the kraft lignin after the different laccase-mediator treatments; (B) The estimated total soluble aromatic compounds in solution determined by Prussian Blue assay; (C) The hierarchical cluster heatmap of the relative abundance of the monomer aromatic compounds in the solubilized lignin solution as detected by GC-MS analysis. Each row of the heatmap represented one detected compound and each column represented one sample (or treatment). The presented values were normalized within the sample and thus represented relative abundance of the compounds. Control: lignin sample without treatment; Lac: lignin sample treated by laccase only; Lac-HBT: lignin sample treated by laccase-HBT; Lac-ABTS: lignin sample treated by laccase-ABTS; Lac-Ace: lignin sample treated by laccase-acetosyringone; Lac-Phenol: lignin sample treated by laccase-phenol. Figure 3. Molecular weight properties of the insoluble fraction of kraft lignin after laccase and laccase-HBT treatments as revealed by GPC analysis. The changes of Mw (A), Mn (B) and PDI (C) for kraft lignin after the laccase-HBT (Lac+HBT) treatment and laccase only (Lac) treatment. Figure 4. Bioconversion of solubilized fraction of kraft lignin out of the laccase-HBT treatment by R.opacus PD630. (A) The growth curve of R.opacus PD630 on solubilized lignin and untreated lignin. (B) The lipid yield of R.opacus PD630 after 7-days fermentation on solubilized lignin and untreated lignin. (C) The heatmap of the abundance of aromatic monomers in solubilized lignin before and after R.opacus PD630 as revealed by GC/MS analysis. The presented values were normalized within the sample.



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Page 26 of 32

Table 1 Consistency (mmol/g) Chemical shift range (ppm)

Assignment Untreated lignin

Lac

Lac+HBT

150.0 – 145.5

Aliphatic OH

2.57

2.33

1.90

144.7---140.0

C5 substituted “condensed”

1.56

1.35

0.98

140.2 – 138.8

Guaiacyl

1.90

1.37

1.10

138.2 – 137.3

p-hydroxylphenyl

0.22

0.21

0.12

136.6 – 133.6

Carboxylic acid OH

0.46

0.32

0.14


Page 27 of 32

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Figure 1



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Figure 2


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Figure 3



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Supporting Information Table S1. The estimated amount of total solubilized aromatic compounds after laccase treatment with different mediators. Table S2. The number-average molecular weights (Mn) and weight-average molecular weights (Mw) of soluble fraction of lignin after enzyme and enzyme-mediator treatments. Table S3. Comparison of the lignin bioconversion efficiency with different lignin depolymerization methods. Figure S1. GPC chromatogram of soluble fractions of control, laccase-treated and laccase-HBT treated lignin. Figure S2. HSQC-NMR spectra (aliphatic regions) of the insoluble fractions of the untreated kraft lignin (Control), laccase treated kraft lignin (Laccase) and laccase-HBT treated kraft lignin (Lac-HBT). Figure S3. The growth curve of R. opacus PD630 on 0.1% (w/v) aromatic model compounds as sole carbon source.



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For Table of Contents Use Only

Advanced Chemical Design for Efficient Lignin Bioconversion Shangxian Xie, Qining Sun, Yunqiao Pu, Furong Lin, Su Sun, Xin Wang, Arthur Ragauskas, and Joshua S. Yuan

Abstract Graphic

Synopsis An efficient enzyme-mediator-microbial system was designed to significantly enhance the electron transfer, lignin depolymerization and bioconversion efficiency.


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Advanced Chemical Design for Efficient Lignin Bioconversion - ACS

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