Pentose fermentation of normally toxic lignocellulose...

Details Pentose fermentation of normally toxic lignocellulose... Pentose fermentation of normally toxic lignocellulose...

2001-06-21

2002-12-24

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S255100, C435S938000

Reexamination Certificate

active

06498029

ABSTRACT:

FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
Effort to produce renewable alternative sources of transportation fuels from biomass have resulted in considerable progress in the conversion of hardwood and agricultural waste into ethanol. Added yields could be expected if the pentosan hemicellulose (five carbon sugar polymers), in addition to hexoses and cellulose, could be effectively fermented to ethanol.
Softwood Prehydrolysates
However, except for sulfite waste liquor, reports of the conversion of softwood materials to ethanol have been limited. Furthermore, the hemicellulose for most softwoods studied is primarily hexosan, composed mainly of mannose with smaller amounts of glucose and galactose, as well as some pentoses. Traditional
Saccharomyces cerevisiae
yeast cultures ferment these hexoses very well, and would be expected to produce high ethanol yields if they could tolerate low concentrations of countless toxins present in dilute acid prehydrolysates generated from softwoods. Typically, these prehydrolysates are generated to produce monomeric sugars from carbohydrate polymers and/or to improve enzymatic digestibility of cellulose in forest waste, municipal solid waste (MSW), or agricultural waste. This biomass is usually converted into a prehydrolysate slurry by soaking the biomass in dilute acid (0.1% to 4%), draining, and then steaming it at about 170° to 215° C. for 30 to 360 seconds.
Prehydrolysates from softwoods are believed to be more toxic than those from agricultural wastes or hardwood biomass sources, because softwoods usually contain more extractives and often more bark than do hardwoods. Consequently, if a process is found to ferment softwood prehydrolysate, fermentation of other prehydrolysates should directly follow. Potential toxic substances include biomass components themselves, particularly extractives such as terpenes, aldehydes, and polyhydroxy aromatics. Other sources of toxins are prehydrolysis products and degradation products including acetic acid from acetylated sugars, furfural, and hydroxy-methyl furfural, the initial degradation products from pentose and hexose sugars, respectively, and oligomers formed by reaction of the furfurals with sugars. Degradation of coniferous lignin yields complex guaiacyl propyl units. Corrosion products from equipment also can be toxic, or the metallic ions can behave as catalysts to produce additional products.
Fortunately most of the toxins in well-prepared prehydrolysate are present at less than one g/L, and only a very few, such as furfural, are present at a few g/L. However, over time, yeast can adapt themselves to tolerate many of these substances in the presence of glucose sugar—but the adaptation in the presence of these toxins prevent or greatly reduce growth and ethanol production.
Yeast Bioreactions and Fermentation
It is generally well known that
Saccharomyces cerevisiae
yeast aerobically oxidizes low concentrations of sugars in aqueous solutions to produce yeast cell mass, carbon dioxide and water, in accordance with the following equation:
1) C
6
H
12
O
6
(~100 g dry wt.)+6O
2
→6 CO
2
+6H
2
O+Cell Mass (~50 g dry wt.)
Under these conditions little or no alcohol is produced. This is how bakers yeast is produced. However, at higher concentrations of sugar, even in the presence of much air, the sugar shuts down the oxidative metabolism of the yeast (the Crabtree effect), and the yeast then ferments the sugars to ethyl alcohol (ethanol), one-third the amount of carbon dioxide, much less cell mass, and no measurable amount of water in accordance with the equation:
2) C
6
H
12
O
6
(~100 g dry wt.)→2 CH
3
CH
2
OH (ethanol, ~51 g)+2CO
2
+Cell Mass (~5 g dry wt.)
This is a forced “fermentation,” with air or without air, because of the Crabtree effect. In the correct sense, fermentation is a term for conversion of sugar to ethanol. Unfortunately, this term has been loosely used for any type of microbial metabolism, even bio-oxidation, by any organism. The loose use of the term fermentation has lead to considerable confusion.
For example, in much of the prior art, even the oxidative conversion of sugars primarily to bakers yeast cell mass is referred to as fermentation.
Prior Art
U.S. Pat. No. 5,693,526 disclose novel strains of yeast
S. cerevisiae
for ethanol production using a number of different hybridization methods for obtaining improved flocculation, improved growth on 40% to 50% molasses (high sugar concentration), and growing in the presence of 7% to 12% ethanol. In this reference, there is no disclosure of hydrolysate, little or nothing on aeration, and little or nothing on low nutrient or on simultaneous aeration, low nutrient, and hydrolysate adaptation.
A process for making yeast tolerant to high pressure in which yeast are transformed with foreign DNA to encode for two enzymes, superoxide dismutase and catalase is disclosed in U.S. Pat. No. 5,674,721. This patent tests the transformed cells to show that they are twice as heat tolerant as the original yeast cells by heating them in the air at about 50° C. The patent does not disclose hydrolysate, evidences little or no discussion on aeration during growth or fermentation, evidences no appreciation for low nutrient or simultaneous aeration, and hydrolysate adaptation.
U.S. Pat. No. 4,567,145 disclose used respiration deficient yeast, and makes no reference to respiration enhanced yeast. This patent does not claim tolerance to hydrolysate nor adaptation to low nutrients. Further, there is no reference in this patent to simultaneous aeration, low nutrient, and hydrolysate adaptation.
Processes for screening aerobic yeast produced from hybridization or mutation using two tests for bread-making which do not use gas-release measurement, such as more growth on maltose, or more growth on sour (acid) dough are disclosed in U.S. Pat. Nos. 4,396,632; 4,318,929 and 4,318,930. These patents generate their yeast by hybridization and mutation, and test these yeast to determine whether they have improved in their performance. These patents do not generate their new strains naturally by simultaneous acclamation under severe constraints over time. Further, the processes in these patents are fully aerobic yeast metabolism, for making bakers yeast. These processes are not fermentation producing ethanol.
U.S. Pat. No. 4,477,569 disclose pentose fermentation by yeast
Pachysolen tannophilus
(not
S. cerevisiae
) with air in the first stage and with improved yield with recycle. The pathway in this process is different and this pathway is not known to exist in
S. cerevisiae
. This patent uses a very rich, very expensive medium including yeast nitrogen base, yeast extract, and casamino acids along with its sugars. Further, this patent states that air is not needed with cell recycling and also uses the mutagen, ethyl methanesulfonate. The patent does not use hydrolysate and does not adapt its cells to tolerate the toxic hydrolysate at very low, inexpensive nutrients simultaneously.
An aerobic fermentation is disclosed for production of high-density yeast cell mass, but not ethanol, in U.S. Pat. No. 4,414,329. The high cell densities are produced in a continuous stirred tank bioreactor by continually feeding mineral salts with the carbon source feed, to eventually achieve cells with high mineral content.
U.S. Pat. No. 3,384,553 disclose a method for aerobic fermentation in which the dissolved oxygen concentration in liquid medium for cultivation of microbes is controlled by the rate of addition of medium to the culture. This patent does not disclose the actual production of ethanol by true fermentation:
“Yeast Adaptation on Softwood Hydrolysate”, in Applied Biochemistry and Biotechnology 70-72, 137-148 (1998) disclose that the highest equivalent total solids (ETS) fermentable hydrolysate concentration is up to 17%, at which concentration we learned that the culture stalled out. In the publication, the concentration of the culture was dying off faster than it was growing, and anything higher than 17% could not be sustained

Affiliated with

Also associated with

Rating

Pentose fermentation of normally toxic lignocellulose... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Pentose fermentation of normally toxic lignocellulose..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Pentose fermentation of normally toxic lignocellulose... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2989418