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Processing biomass

a technology of biomass and enzymes, applied in the field of processing enzymes, can solve the problems of low yield of naturally occurring cellulosic materials, low yield of complex matrix, and high cost of natural-occurring cellulosic materials,

Inactive Publication Date: 2014-01-09
XYLECO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes methods and compositions for inducing the production of enzymes by a microorganism using a cellulosic or lignocellulosic biomass that has been treated to reduce its recalcitrance. The treatment can include any of a variety of methods such as sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, and freeze grinding. The biomass can be any of a variety of materials such as paper, paper products, paper waste, and forestry wastes. The microorganism can be a fungus, bacterium, or yeast, and can be genetically engineered or a strain that produces high levels of cellulase. The methods and compositions can also include removing all or a portion of the liquid from the microorganism-inductant-enzyme combination, concentrating or isolating the enzymes, and allowing saccharification of the biomass to occur. The technical effects of this invention include improved efficiency in the production of enzymes for saccharification of cellulosic or lignocellulosic biomass and the development of new methods for reducing the recalcitrance of biomass.

Problems solved by technology

This compact matrix is difficult to access by enzymes and other chemical, biochemical and biological processes.
Cellulosic biomass materials (e.g., biomass material from which substantially all the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, naturally-occurring cellulosic materials often have low yields (relative to theoretical yields) when contacted with hydrolyzing enzymes.
While a number of methods have been tried to extract structural carbohydrates from lignocellulosic biomass, they are either too expensive, produce too low a yield, leave undesirable chemicals in the resulting product, or simply degrade the sugars.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cellulase Performance Test on Paper, Treated Corn Cob and Untreated Corn Cob

[0151]Various inducers (treated biomass (TBM), untreated biomass (UBM), paper (P) and carboxylmethylcellulose (CMC, Aldrich) ) were used to produce enzymes. The biomass (TBM and UBM) was milled corn cob collected between mesh sizes of 15 and 40. Treatment of the biomass (UBM) to produce the TBM involved electron bombardment with an electron beam to a total dose of 35 Mrad. The paper was shredded and screened to have a nominal particle size smaller than 0.16 inch. The inducer experiments were conducted using shake flasks and PC3-7 and RUT-C30 strains. After 3 days of the growth culture, the harvested cell mass was added to a series of shake flasks each containing 50 ml of medium and 1 wt. % of one of the inducers.

[0152]The induction experiment was allowed to proceed for 11 days. The culture supernatant was then harvested by centrifugation at 14,500 rpm for 5 minutes, and stored at 4° C.

[0153]Protein concentra...

example 2

Enzyme Production in Different Concentrations of TBM Inducer

[0156]This Example was done using bioreactors. Cell strain RUT-C30 was propagated in the media with 2.5% glucose. After 3 days of growth the culture was centrifuged and the cells were re-suspended in 50 ml of media with 1, 3, 5, 7 and 9 wt. % TBM. The protein concentrations and activities after 11 days of incubation at 27° C. and 200 rpm are shown in the table below.

TABLE 2Amounts of Protein and Enzyme MadeWith Differing Amounts of InducerInducer amountProteinFPUCMC(wt. %)(g / L)(U / mL)(U / mL)10.71.41.321.43.11.753.46.22.672.92.51.591.50.61.0

[0157]These results show that higher levels of enzymes were produced when the treated biomass (TBM) was added at a rate of 5%.

[0158]Saccharification of biomass with enzymes: Saccharification of biomass (TBM) using enzymes produced by addition of 2, 5 and 7 wt. % treated biomass inducer (TBM) versus a commercial enzyme (Duet™ Accellerase, Genencor) was conducted. The biomass, 10 wt. % TBM, w...

example 3

SDS-PAGE of Enzyme Produced With Treated Biomass

[0159]A bioreactor culture was prepared using the method described above except that the mixing was done at 50 rpm rather than 500 rpm. The protein assay showed that 3.4 g / L protein was produced.

[0160]The analysis of the protein using SDS PAGE is shown in FIG. 5. Lane 1 and 5 are molecular weight markers, Lane 2 is a 30 uL load of the protein, Lane 3 is a 40 uL load of the protein, Lane 4 is Duet™ Accelerase enzyme complex (Genencor).

Example 4. Range of Conditions TestedParametersRangeWorkingBestInductionTestedTestedRangeRangeCorn CobParticle Size1400-1400-Amount Added25-133 g / L25-133 g / L100 g / LTiming ofDay 0-31-3Day 1-3AdditionFrequency of1, 2, and 51, 2, and 51AdditionPresentationwet or drywet or drywet or dryTreatment Levels353535LactoseTiming ofDay 3Day 3Day 3AdditionAmount Added4.7-40 g / L / d4.7-18.7 g / L / d18.7 g / L / dFrequency ofcontinuouscontinuouscontinuousAdditionfeedfeedfeed

[0161]Any patent, publication, or other disclosure materi...

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Abstract

Provided are methods of inducing enzymes, and for treating cellulosic and lignocellulosic biomass with the enzyme.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of PCT / US2012 / 071091, filed Dec. 20, 2012, which claimed priority from U.S. Provisional Application Nos. 61 / 579,550 and 61 / 579,562, both filed on Dec. 22, 2011. The entire disclosures of the above applications are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention pertains to the preparation enzymes useful in the processing of biomass materials. For example, the invention relates to producing cellulase enzymes or other enzyme types.BACKGROUND[0003]As demand for petroleum increases, so too does interest in renewable feedstocks for manufacturing biofuels and biochemicals. The use of lignocellulosic biomass as a feedstock for such manufacturing processes has been studied since the 1970s. Lignocellulosic biomass is attractive because it is abundant, renewable, domestically produced, and does not compete with food industry uses.[0004]Many potential lignocellulosic feedstocks are availa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/42
CPCC12N9/42C12N9/2434C12N9/2437C12P5/02C12P5/023C12P7/04C12P7/10C12P7/16C12P7/24C12P7/28C12P7/40C12P7/42C12P7/46C12P7/48C12P7/52C12P7/56C12P7/62C12P19/02C12P19/04C12P19/12C12P19/14C12Y302/01004C12P2201/00Y02E50/10Y02E50/30
Inventor MEDOFF, MARSHALLMASTERMAN, THOMAS CRAIGYOSHIDA, AIICHIROMOON YEE FUNG, JENNIFERLYNCH, JAMES J.
Owner XYLECO INC
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