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Alcohol, Protein and Vinegar Production |
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By Steve D. Wilson, Ph.D. e mail to
wri2@juno.com |
I.
INDUSTRIAL AND AGRICULTURAL WASTE RECOVERY
A. DEHALOGENATION OF INDUSTRIAL AND AGRICULTURAL WASTE
1. BACKGROUND
The problem of environmental contamination by toxic and/or carcinogenic halogenated hydrocarbons (HHs) is a worldwide critical problem whose
solution is wanting. According to the EPA there are 114 organic chemicals established as priority pollutants contaminating ground water, surface
water, soil, and air.
2. TREATMENT
Techniques for treatment of contaminated water include the drilling of new wells, aeration and activated charcoal treatment of the contaminated water. Unfortunately HHs still exists and must ultimately be released to the atmosphere.
A solution to solve HHs is by biological decomposition and to utilize several methanogenic bacteria so as to degrade HHs to produce ethane,
ethylene, and acetylene as by-products.
The particular organisms we are developing are Methanococcus thermolithotrophicus, Methanobacterium thermoautotrophicum, and Methanococcus deltae.
B. ALCOHOL FROM AGRICULTURAL WASTE AND OTHER POTENTIAL CROPS
1. BACKGROUND
Fermented agricultural feed stock, particularly that of grains and fruit to yield ethanol alcohol, has been produced for centuries (one suspects that
even early man in the predawn of communicable languages, knew about fermentation).
In more modern times, ethanol alcohol has been used for fuel and this production from the fermentation of agricultural crops and waste is strongly supported by the farming community. The public has a greater awareness in the need to augment our fuel sources and has a favorable view in using
ethanol alcohol as an alternative automotive fuel. Furthermore the federal government, as well as state and local governments, have recognized the potential for ethanol as a fuel additive or substitute and indeed have responded by various positive ways (reduction of excise tax, grants, public
awareness, etc.). Because of these encouragements, ethanol production increased from a few million gallons to over 50 million gallons annually.
Most of the production was blended to obtain gasohol.
Under public law 96-294 Congress established production criteria of at least a billion gallons as an annual production. Additionally there exist
markets for ethanol in the pharmaceutical industries; for pure medical alcohol; and for industrial use such as conversion to acetic acid and ethylene.
2. METHOD
After considerable research and study to determine the enzymes and yeasts best suited for the fermentation of sugar bearing agricultural products
and/or agricultural waste products, a pilot plant was set up at Del Mar Foods Watsonville, CA.
The feedstock entering the plant was dumped into large hoppers. Sample tests for sugar were made and then the feedstock was screw-fed through a hammer mill and passed directly into fermentation tanks.
Some feedstock (squeezed apple waste) was rehydrated with other sugar containing liquid waste. Yeasts of a specific type and nutrients were added
as soon as the product entered the tank. The pH was balanced for optimum fermentation and the temperature was monitored. The tanks were then capped. One-way valves allowed carbon dioxide and other gases to be released while the batch was intermittently agitated to maintain uniform fermentation.
The tanks contained thermostatically operated heat exchangers that automatically maintained the brewing temperature. Excess heat was used to
preheat the next batch tank.
In this manner fermentation periods were reduced by 75% with alcohol yields of 3% to 4.5%. Distillation was performed in the laboratory with results indicating that the alcohol and feed potential was as originally projected. Approximately 1,270 gallons of waste apples containing 11.4% sugar content yielded 50.75 gallons of 180 proof (90%) ethanol alcohol. Other by-products that came from the fermentation process were high protein animal feed.
After fermentation the mash was pumped and/or screw fed into the vaporization kettle where heat was applied to drive the alcohol vapor into the
distillation tower. Ethanol, in the range of 180 proof, was drawn off and stored in a fireproof tank.
The remaining mash was pressed to remove up to 70% of its moisture in the form of stillage, which contained acids and esters. The remaining solids
were air-dried.
3. MARKETABLE PRODUCTS
We are capable of producing, by contract fermentation, the hybrid yeast that has a higher percentage yield and diminishes the fermentation time or
put into operation an entire processing plant.
There are potentially five marketable products; ethanol, beer, run-off, stillage, and solids. By definition:
BEER: This is the liquid solution that results from the fermentation process.
ETHANOL: C2 H5 OH is the alcohol product of fermentation of sugar and extracted from the beer by distillation.
RUN-OFF: This is the liquid by-product of distillation that remains after the alcohol has been drained off.
STILLAGE: The "dregs" that remain in the distillation kettles after all alcohol has been vaporized.
SOLIDS: The leftover residue remaining after the stillage
has been pressed out.
Alcohol Fermentation Experimental Results
Batch DM-l constituted
waste apple from Del Mar Food Products. Fermentation began April 29th with
total weight of 16 lbs. One month later the sugar content was 5.7%.
This indicates 20.27 ounces of sugar by weight of which 5.7% sugar remained after fermentation leaving 48% or 9.73 ounces of sugar
supposedly converted
to 200 proof alcohol. Actual recovery after distillation
of the total volume available. This is not unusual
expect approximately
This batch had 9.73 oz of sugar available and 45% of that yield would be 4.38 oz. of alcohol. We got approximately the 45% or 8.30 oz. of
100
proof alcohol after some loss in transfer due to gasket leakage in the still.
The recovery is 5% of the total fermented liquid by weight or 3.2% of
the total solid weight of the original sample.
Batch DM-2 consisted of waste apple from Del Mar Foods with sugar added- to push the sugar content to 19%.
After a month of
fermentation, we extracted 9 lbs. of fermented liquid for distillation.
The sugar content was reduced-to 4.8% yielding 20.45 ounces of sugar involved in the alcohol conversion process.
At 45% conversion efficiency this should yield 9.2 ounces of 200 proof alcohol.
We recovered 5.8 ounces of 140 proof alcohol or 28% of the available fermented sugar. A large amount was lost in the distillation process.
This, however, is still a 4% recovery of the total fermented liquid.
Batch DM-3 consisted
of 20 lbs. of Del Mar waste apple with 13.3% initial sugar content. Fermentation was begun on
2
grams of No. 3 yeast. By May 31st, after three days of fermentation,
sugar content was 6.3%.
which
yielded 4 oz. of 85 proof alcohol.
In this case we had 7.42 oz. of sugar available, which after 45% conversion efficiency should yield 3.34 oz of 200 proof alcohol. Our actual
recovery
was 4% of the total fermented liquid by weight.
Batch DM-4 consisted of 19 lbs. of Del Mar apple waste with 13.3% initial sugar content. This yielded 186 fluid ounces of the material. After
three days of fermentation using No. 4 yeast, 6.3 ounces of 70 proof alcohol was recovered. This was considerably less than the equivalent
of
5.58 fluid ounces of 200 proof alcohol that was possible. Losses are attributed
to vapor seepage around the gasket of the still.
Batch DM-5 consisted of waste apple mash from Del Mar Food Products. Initial sugar content was 10.5%. This batch was preheated and treated
with nitrogen rich nutrients and the pH was stabilized
at 4.0. Fermentation was completed in three days using
yeast No.3 and a variation on the
classical approach of fermentation.
Juice extractors were used to recover the maximum liquid content from the samples. Out of the total combined solid liquid weight of 13 pounds,
8 lbs. 4 oz. of distillate liquid was recovered.
From this liquid 3
ounces of 132 proof and 10.2 ounces of 70 proof alcohol was extracted.
This is equivalent to 5.6 ounces of 200 proof (pure) alcohol which, constitutes 4.2% of the total distillable liquid by weight.
Batch DM-6 consisted of Del Mar waste apple mash. This batch was similarly preheated as in Batch DM-5. Yeast No. 4 was used for
fermentation and the same nitrogen rich nutrients were added. pH was stabilized at 4.0. After three days of fermentation, juice extractors were
used to recover the maximum liquid content from the mash. of 13 pounds, From the total original weight of the sample 6 lbs. 2 oz. of distillable
liquid was
extracted.
From this liquid, 5
ounces of 70 proof and 6 ounces of 40 proof alcohol was extracted. This is
equivalent to 3 ounces of 200 proof
alcohol which constitutes 3.06% of the total distillable liquid by weight.
alcohol yield was lost due to inefficiency of the still available to us.
Batch DM-6, for example, had an original sugar content of 10.5%. After fermentation, sugar content was at 3.8% indicating 6.7% of the sugar was
acted upon. Out of this 6.7%, 45% was converted to alcohol or 3% of the total original batch by weight. 155 total ounces should have yielded 4.67 ounces of 200 proof alcohol.
We extracted an equivalent of 3 ounces of 200 proof alcohol indicating a loss of 1.67 ounces. This loss is equivalent to a 36% loss of the originally available alcohol by weight.
We conclude that even under less than optimum conditions, we have extracted enough alcohol from the Del Mar waste stream in a timely enough
manner to prove the economic feasibility of this part of the operation.
We have succeeded in reducing fermentation time from one month to three days after researching particular yeasts and enzymes that can work
efficiently with apples. Also, we feel that developing a column fractional distillation process of more or less conventional design would be sufficient
to extract nearly all available alcohol thereby increasing cost effectiveness considerably.
As of now, our original cost benefit projections are proving valid. We will, in the next series of experiments, refine the batch fermentation formulation
for higher yields and make an effort to stop leakage in the still.
If tests being conducted simultaneously on the nutritional and soil conditioning values of pre and post distillation by-products prove out, our test will
be judged successful and a large scale operation should begin as soon as possible.
Steve D. Wilson, Ph.D.
Western Research Institute
Appendix A
WHEY
INTO PROTEIN
CONVERSION OF WHEY, PRIMARILY A WASTE PRODUCT, INTO USEFUL BY-PRODUCTS
1. INTRODUCTION
Whey is a waste product of cheese making and the composition of whey varies with the type of cheese produced. But basically whey is composed of
4 to 5 % lactose, a disaccharide, 90% water, 0.80% protein, 5 to 6% solids, and some minerals and ash. Approximately 60% of the whey in the
USA is utilized
the remaining 40% is discharged down sewage lines or dumped at fill sites. With
the recent aggressive clean air and water act throughout the USA and
particularly in California, cheese-producing companies are seeking new
solutions to converting whey into useful and salable by-products.
Some of the by-products are:
1. A
glucose/galactose syrup.
2. A low calorie
high nutritional value drink.
3. A high protein
food.
4. Cattle feed
mixed with algae and hyacinth.
5. Alcohol.
6. Reusable water.
7. Yeast.
WRI Inc. has done extensive research into the production of alcohol and/or high grade protein from whey. A pilot plant was set up in Hanford, CA
and a batch fermentation process was carried out with protein yield as the
first goal.
2. METHOD
We initially filled an open tank with 150 gallons (570 L) of whey and, through solar distillation, diminished the volume to 112 gallons (425 L) or by
25% in a single pass (centrifugation would be necessary if solar distillation is not used). We stabilized the pH to 6.5 by introducing calcium hydroxide
and cooled the batch to 104 degrees F (40 degrees
C).
At this point we introduced 0.6 qt. (570ml) of a lacto-enzyme (1500 LAU/L concentration). The reaction time was 30 minutes to convert the lactose (disaccharide) into glucose and beta-galactose. We further cooled the converted batch to 86 degrees F (30 degrees C) brought the pH down to 4.5
by adding phosphoric acid and introduced our special yeast (Saccharomyces cerevisiaex1)(1/4lb.) to convert the sugars into more yeast instead of alcohol. We drained off the liquid into a holding pond where we introduced algae and water hyacinth so as to remove the nutrients and heavy metals
from the water. The remaining yeast
in the batch tank (62% protein) was air dried and bagged.
In place of the microorganism S. cerevisiae one could use a strain of Kluyveromyces fragilis that has a higher yield of whey to
alcohol and requires a two-stage continuous batch fermentor to be efficient.
Biotechnology
Yeast (Saccharomyces cerevisiae) especially hybridized to convert whey or whey permeate into a high protein food supplement for man or beast.
Careful balance of pH and temperature in an aerobic environment throughout the conversion of the disaccharide (lactose) into the simpler sugars by
the addition of the enzyme B-galactosidase and subsequently adding the special yeast under favorable pH, temperature and proper aeration to
produce a 50% to 75% protein mix.
A variation of the same yeast under proper conditions becomes a rapid fermentor of sugar containing waste products yielding alcohol. We then use the solar distiller to produce 180 proof alcohol. The solids are dried in the sun and used for animal food or dried in a solar dryer (Appendix A).
\
Vinegar
Production from Agricultural waste
A. INTRODUCTION
Preliminary feasibility study of high grain vinegar production through the control and selection of specialized aceti organisms.
Natural vinegar is a liquid containing the acetic acid obtained by the acetous fermentation or by oxidation of alcohol liquids. Vinegar obtained by
acetous fermentation possesses properties and substances such as volatile oils, compound ethers, esters, acids, ketones, and dissolved solids
rendering a color varying from pale yellow to deep
brownish red. Its odor, taste and color are influenced by the materials
employed in its manufacture.
Synthetic vinegar or distilled vinegar, prepared from commercial acetic acid lacks the bouquet bodies found in natural vinegar, therefore, the objective
of this study is to see if it is feasible to produce 120 grain or more of natural apple vinegar.
A variety of techniques are employed in making vinegar, that is, by the slow process, the quick process, the use of graduators, or the continuous thin
plate generator. Our concern is not on what process to employ but whether the aceti organisms can survive in up to 14% alcohol and consistently
produce 120 to 140 grain vinegar cider containing the desired bodies. Our initial process will be to examine a variety of aceti organisms such as
mycoderms aceti , acetobacter aceti and possibly A. xylinum, in a variety of controlled liquids and selection so as to arrive at an organism that
will produce the end results we desire.
B. Isolation of Acetobacter suboxydans mutants which yields higher concentrations of acetic acid.
1. Backround and rationale
A. aceti can
be used to yield higher levels of acetic acid via the isolation of spontaneous
mutants capable of
tolerating
increased acetic acid concentration, or decreasing pH.
However, A.
aceti is an over-oxidizer, converting glucose--.ethanol--acetic
acid --CO2 + H2O..
The oxidization of acetic acid to CO2 and H2O limits the desirability of A. aceti unless considerable care is taken to avoid this reaction.
An alternative to this problem is the substitution of A. suboxvdans. The species is an under-oxidizer in that it takes ethanol almost stoichiometrically to acetic acid, which it is wholly incapable of further attacking.
The production of acetic acid by A. suboxvdans is slower than that observed with A.aceti, by a factor of about 1.7.
However, the
advantage of under-oxidation can be further amplified by isolating mutants of A.
suboxvdans
possess decreased generation times; as well as mutants capable of
withstanding decreased pH, resulting from higher concentrations of acetic
acid.
Such mutations
are possible and result from molecular mechanisms involving alterations in
the genetic makeup (DNA) of
the cell. Thus, it is possible. to induce point mutations, deletions,
additions and substitutions, to name a few, in the cell's DNA and to screen
the surviving cell population for cells possessing the desired
characteristics, i.e, rapid growth and acid tolerance in the case of A.
suboxvdans.
C. Materials
and methods required to isolate A. suboxvdans mutants.
Nitrosoguanidine (NS:;)
is a powerful mutagen, which has been
used in my laboratory for several years. It reacts at both the DNA and RNA
levels, producing multiple mutations.
Surviving mutants are easily recognized by colonial morphology on agar plates.
Typically, a nutrient broth culture of A.suboxvdans will by treated at an interval of time with NG.
The treated, sample is then diluted, to reduce the concentration and avoid further reaction, 'then added to fresh broth to allow mutational expression at about 37°C for 1-2 days.
The culture is then diluted and a small volume (0-1 ml) is spread onto a nutrient agar plate(s). After incubating the plates for 1-2 days at 37°C the
colonies are counted and examined for differences in colonial morphology, i.e. size, sectoring, texture, etc. Using sterile tooth picks, a portion of each colony is inoculated into nutrient broth containing l-l.5% glucose. Inoculated tubes are incubated for 1-2 days at 37 °C and subsequently tested for
acetic acid concentrations via standard chemical analysis.
An alternative
method for screening very large (>100,000) number of colonies is the use of
velveteen replicating
D.
Estimated time required for this project.
Since the gene pool of the bacterium A. suboxydans is probably equal to if not greater than that of other wall characterized bacteria, i.e. E. coli, B. subtilis etc. and this should be greater than: 5000 genes, the number of genes involved in acetic acid production may be 12 and the number involved
in cell division may be 50, since many interrelating genes are involved, the isolation of the particular class of mutant desired will require examination
of an as yet
unknown number of mutants. However, it is anticipated that no more than
500,000 isolates will be examined.
The time required for such an examination and analysis of acetic levels is estimated to be approximately three weeks.
E.
Summary
A. suboxydans will be mutaginized with N.G. Surviving cell populations will be examined for mutations affecting generation time and acetic production.
It is anticipated that based upon known mutagenic mechanisms N.G. should produce the desired mutants. It is further thought that no more than
500,000 colonies
need be examined within approximately a three week period before the project
is terminated. However, based upon all available information, the project has a high
probability of success.