Thermochemical wastewater valorization: Via enhanced microbial toxicity tolerance

Lahiru N. Jayakody, Christopher W. Johnson, Jason M. Whitham, Richard J. Giannone, Brenna A. Black, Nicholas S. Cleveland, Dawn M. Klingeman, William E. Michener, Jessica L. Olstad, Derek R. Vardon, Robert C. Brown, Steven D. Brown, Robert L. Hettich, Adam M. Guss, Gregg T. Beckham

Research output: Contribution to journalArticlepeer-review

83 Scopus citations

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. Overall, 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.

Original languageEnglish
Pages (from-to)1625-1638
Number of pages14
JournalEnergy and Environmental Science
Volume11
Issue number6
DOIs
StatePublished - Jun 2018

Funding

The NREL authors thank the US Department of Energy (DOE) Energy Efficiency and Renewable Energy (EERE) Bioenergy Technologies Office (BETO) for funding this work via Contract No. DE-AC36-08GO28308. JMW, RJG, SDB, RLH, AMG, and GTB acknowledge funding for the multi-omics efforts by the Bio-Energy Science (BESC) and the Center for Bioenergy Innovation (CBI), both U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. Transcriptomics data were generated by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. We thank Mary Biddy, Abhijit Dutta, Christopher Kinchin, Josh Schaidle, and other NREL colleagues for helpful conversations regarding pyrolysis wastewater and Anna Knapp, Payal Khanna, and Emily Fulk for their assistance in constructing the pK18sB vector. We thank David Dayton at RTI International for providing pyrolysis-derived wastewater streams. We thank Víctor de Lorenzo at the Centro Nacional de Biotecnología (CNB-CSIC) for providing the P. putida EM42 strain. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

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