Adaptive laboratory evolution of nanocellulose-producing bacterium

Authors: VASCONCELLOS, VANESSA - Federal University - Brazil; FARINAS, CRISTIANE - Federal University - Brazil; XIMENES, EDUARDO - Purdue University; Slininger, Patricia - Pat; LADISCH, MICHAEL - Purdue University

Submitted to: Biotechnology and Bioengineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2019
Publication Date: 4/30/2019
Citation: Vasconcellos, V.M., Farinas, C.S., Ximenes, E., Slininger, P., Ladisch, M. 2019. Adaptive laboratory evolution of nanocellulose-producing bacterium. Biotechnology and Bioengineering. 116(8):1923-1933. https://doi.org/10.1002/bit.26997.
DOI: https://doi.org/10.1002/bit.26997

Interpretive Summary: Liquid hot water pretreatment of lignocellulosic materials including corn stover, corn fiber, switchgrass, sugarcane and hardwoods opens cellulosic fibers for saccharification but also results in an aqueous liquor byproduct stream that is low in glucose sugar concentration and abundant in inhibitory acetic acid and phenolic constituents. Gluconoacetobacter species could be used to valorize this stream by directly converting, and thereby concentrating, the glucose present into an insoluble, readily recovered value-added product called nanocellulose. However, inhibitor tolerant strains of the bacteria are required to avoid the cost to detoxify this pretreatment liquor prior to bioconversion. Adaptative evolution was applied in the laboratory by exposing G. Hansenii to increasing concentrations of pretreatment liquid from corn stover. The result was a resistant strain of G. hansenii that generated bacterial nanocellulose directly from pretreatment liquids of corn stover without need for prior detoxification steps to remove inhibitors. As a result of this strain improvement, the low value pretreatment liquor has become a valuable resource for production of bacterial nanocellulose with potential applications in food areas, including hydrocolloid additives and packaging, and in medical areas for wound dressings, tissue replacement, and drug delivery. This research directly supports a developing bioeconomy utilizing agricultural products.

Technical Abstract: Adaptive laboratory evolution through 12 generations of the nanocellulose producing bacterium Gluconoacetobacter hansenii (ATCC 23769) in liquid filtrate from hydrothermal pretreatment of corn stover resulted in a strain that resists inhibition by phenolics in pretreatment liquid. The original strain generated nanocellulose from glucose in standard Hestrin and Schramm (HS) medium, but not from the glucose in pretreatment liquid. G. hansenii cultured in pretreatment liquid treated with activated charcoal to remove inhibitors also converted glucose to bacterial nanocellulose. The properties of this cellulose were the same as nanocellulose generated from media specifically formulated for bacterial cellulose formation. However, attempts to directly utilize glucose proved unsuccessful due to the toxic character of the lignin derived phenolics, and in particular, vanillan and ferulic acid. Adaptative laboratory evolution at increasing concentrations of pretreatment liquid from corn stover in HS medium resulted in a strain of G. hansenii that generated bacterial nanocellulose directly from pretreatment liquids of corn stover. The development of this adapted strain positions pretreatment liquid as a valuable resource since G. hansenii is able to convert and thereby concentrate a dilute form of glucose into an insoluble, readily recovered and value-added product – bacterial nanocellulose..