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Title: Thermochemical wastewater valorization via enhanced microbial toxicity tolerance

Abstract

Thermochemical (TC) biomass conversion processes such as pyrolysis and liquefaction generate considerable amounts of wastewater, which often contains highly toxic compounds that are incredibly challenging to convert via standard wastewater treatment approaches such as anaerobic digestion. These streams represent a cost for TC biorefineries, and a potential valorization opportunity, if effective conversion methods are developed. The primary challenge hindering microbial conversion of TC wastewater is toxicity. In this study, we employ a robust bacterium, Pseudomonas putida, with TC wastewater streams to demonstrate that aldehydes are the most inhibitory compounds in these streams. Proteomics, transcriptomics, and fluorescence-based immunoassays of P. putida grown in a representative wastewater stream indicate that stress results from protein damage, which we hypothesize is a primary toxicity mechanism. Constitutive overexpression of the chaperone genes, groEL, groES, and clpB, in a genome-reduced P. putida strain improves the tolerance towards multiple TC wastewater samples up to 200-fold. Moreover, the concentration ranges of TC wastewater are industrially relevant for further bioprocess development for all wastewater streams examined here, representing different TC process configurations. Furthermore, we demonstrate proof-of-concept polyhydroxyalkanoate production from the usable carbon in an exemplary TC wastewater stream. Altogether, this study demonstrates that protein quality control machinery and repairmore » mechanisms can enable substantial gains in microbial tolerance to highly toxic substrates, including heterogeneous waste streams. When coupled to other metabolic engineering advances such as expanded substrate utilization and enhanced product accumulation, this study generally enables new strategies for biological conversion of highly-toxic, organic-rich wastewater via engineered aerobic monocultures or designer consortia.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [2];  [1];  [1]; ORCiD logo [2];  [1];  [1]; ORCiD logo [1]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Iowa State Univ., Ames, IA (United States)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); LanzaTech, Inc., Skokie, IL (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1542241
Alternate Identifier(s):
OSTI ID: 1434804
Report Number(s):
[NREL/JA-2A00-71485]
[Journal ID: ISSN 1754-5692; EESNBY]
Grant/Contract Number:  
[AC05-00OR22725; AC36-08GO28308; AC02-05CH11231]
Resource Type:
Accepted Manuscript
Journal Name:
Energy & Environmental Science
Additional Journal Information:
[ Journal Volume: 11; Journal Issue: 6]; Journal ID: ISSN 1754-5692
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS

Citation Formats

Jayakody, Lahiru N., Johnson, Christopher W., Whitham, Jason M., Giannone, Richard J., Black, Brenna A., Cleveland, Nicholas S., Klingeman, Dawn Marie, Michener, William E., Olstad, Jessica L., Vardon, Derek R., Brown, Robert C., Brown, Steven D., Hettich, Robert L., Guss, Adam M., and Beckham, Gregg T. Thermochemical wastewater valorization via enhanced microbial toxicity tolerance. United States: N. p., 2018. Web. doi:10.1039/c8ee00460a.
Jayakody, Lahiru N., Johnson, Christopher W., Whitham, Jason M., Giannone, Richard J., Black, Brenna A., Cleveland, Nicholas S., Klingeman, Dawn Marie, Michener, William E., Olstad, Jessica L., Vardon, Derek R., Brown, Robert C., Brown, Steven D., Hettich, Robert L., Guss, Adam M., & Beckham, Gregg T. Thermochemical wastewater valorization via enhanced microbial toxicity tolerance. United States. doi:10.1039/c8ee00460a.
Jayakody, Lahiru N., Johnson, Christopher W., Whitham, Jason M., Giannone, Richard J., Black, Brenna A., Cleveland, Nicholas S., Klingeman, Dawn Marie, Michener, William E., Olstad, Jessica L., Vardon, Derek R., Brown, Robert C., Brown, Steven D., Hettich, Robert L., Guss, Adam M., and Beckham, Gregg T. Tue . "Thermochemical wastewater valorization via enhanced microbial toxicity tolerance". United States. doi:10.1039/c8ee00460a. https://www.osti.gov/servlets/purl/1542241.
@article{osti_1542241,
title = {Thermochemical wastewater valorization via enhanced microbial toxicity tolerance},
author = {Jayakody, Lahiru N. and Johnson, Christopher W. and Whitham, Jason M. and Giannone, Richard J. and Black, Brenna A. and Cleveland, Nicholas S. and Klingeman, Dawn Marie and Michener, William E. and Olstad, Jessica L. and Vardon, Derek R. and Brown, Robert C. and Brown, Steven D. and Hettich, Robert L. and Guss, Adam M. and Beckham, Gregg T.},
abstractNote = {Thermochemical (TC) biomass conversion processes such as pyrolysis and liquefaction generate considerable amounts of wastewater, which often contains highly toxic compounds that are incredibly challenging to convert via standard wastewater treatment approaches such as anaerobic digestion. These streams represent a cost for TC biorefineries, and a potential valorization opportunity, if effective conversion methods are developed. The primary challenge hindering microbial conversion of TC wastewater is toxicity. In this study, we employ a robust bacterium, Pseudomonas putida, with TC wastewater streams to demonstrate that aldehydes are the most inhibitory compounds in these streams. Proteomics, transcriptomics, and fluorescence-based immunoassays of P. putida grown in a representative wastewater stream indicate that stress results from protein damage, which we hypothesize is a primary toxicity mechanism. Constitutive overexpression of the chaperone genes, groEL, groES, and clpB, in a genome-reduced P. putida strain improves the tolerance towards multiple TC wastewater samples up to 200-fold. Moreover, the concentration ranges of TC wastewater are industrially relevant for further bioprocess development for all wastewater streams examined here, representing different TC process configurations. Furthermore, we demonstrate proof-of-concept polyhydroxyalkanoate production from the usable carbon in an exemplary TC wastewater stream. Altogether, this study demonstrates that protein quality control machinery and repair mechanisms can enable substantial gains in microbial tolerance to highly toxic substrates, including heterogeneous waste streams. When coupled to other metabolic engineering advances such as expanded substrate utilization and enhanced product accumulation, this study generally enables new strategies for biological conversion of highly-toxic, organic-rich wastewater via engineered aerobic monocultures or designer consortia.},
doi = {10.1039/c8ee00460a},
journal = {Energy & Environmental Science},
number = [6],
volume = [11],
place = {United States},
year = {2018},
month = {4}
}

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Figures / Tables:

Figure 1 Figure 1: Baseline toxicity of FPF stream component to toxicity with P. putida KT2440. (A) Growth rate of P. putida in the FPF stream as a function of dilution factor. (B) Growth rates of P. putida KT2440 in M9 medium containing 20 mM glucose and different concentration of synthetic FPFmore » (FPFSYN) or actual FPF (FPFACT). (C) The effect of functional group classes in the FPF stream on the growth of P. putida in minimal (M9) media containing 20 mM glucose. The medium was supplemented with a combination of compounds belonging to the functional groups at the same concentration found in a 0.1% (v/v) dilution of the FPF stream, which is a lethal concentration to P. putida KT2440. (D) Contribution of different functional groups to the toxicity of the FPF stream on P. putida KT2440. Results are expressed as means ± SEM (n=3). Bars labeled with different symbols ( $\alpha,\beta,\varepsilon$ ) indicate statistical significance of the functional groups (p < 0.05; one-way ANOVA followed by Tukey’s post hoc honest significance difference test). Bars labeled with the same symbol indicate no statistically significant difference (p > 0.05). FPF: fast pyrolysis with-fractionation.« less

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