Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to a process for preparing a
biomass. More particularly, the invention relates to a process
whereby a rnixture containing fermentable materials, such as
carbohydrates, more particularly sulfite waste liquor, is
allowed to be bio-degraded with microorganisms derived from
domestic sewage to give a biomass which can he used for feed-
ing animalsO
Small bisulfite and siulfite mills are known for their
water pollution problems. These mills normally produce less
than 300 tons per day of pulpo ~le water pollution load in
the effluents from these mills results mainly from the wood
carbohydrates dissolved in the waste pulping liquors-which
are presently discharged to the receiving water~.
On the other hand, the various governments are soon
due to implement water pollution regulations for pulp and
paper industries. These regulations will be so stringent in
the very near future that mills wi]l have to treat their
effluents. For exarnple, the Quebec Government's
water pollution regulation for bisulfite pulp mills, which
will come into force by the end of 1980, will require
that an existing mill mus~ remove 65% of BOD5 in its combined
ef~luent before discharging. ~ecause of their small capaci~y
in pulp production, mosit mills cannot economically adopt the
conventional treatment system to recover cheMicals ancl heat
from their waste liquors, asi most kra~t mill~ do at present.
~he biomass derived from pure cul~ure such as torula yeast
produced from waste sulfite liquor, has long been used in hu-
man food~ [Scrimshaw, N.S., "Single-Cell Protein" ed.: Mateles,
R.I. and Tannenbaurn, S.R., P. 5, MIT Press, Cambridge, Mass.
(1968)] and animal feed [Peppler, J.H., "Single-Cell Protein",
ed.: Mateles, R.I. and Tannenbaurn, S.R., p. 238, MIT Press
Cambridge, Mass. (1968)~.
It is also well known that feeds used for animals can
be replaced totally or partially with pure culture biomass
(microfungi) resulting from the bio-degradation of different
sulfite waste liquors. All these processes require careful
preparation and purification of the microorganisms. Also, the
biological reaction and the other operations must be carried
out under rigid control and aseptic conditions, in order to
prevent external contamination.
It i5 also well known that throughout the world there
is a shortage of animal feeds and since this shortage problem
should become more serious in the future, it would seem inter-
esting to solve this problem, at least partially, by using as
a portion of the feed, a biomass resulting from the bio-degra-
dation of waste pulping liquors, especially where the pulp and
paper industry produces an extremely large amount of waste li-
quor. I~his shortage of animal feed and the excess of waste
pulping llquors are especially true for Eastern Canada.
With this view in mind, the present invention aims at
a process for preparing a biomass which comprises:
a) providing liquor containing refuse fermentable
materials;
b~ providing a reactor containing microorganisms
derived from sewage;
c) rnixing said liquor with nutrients,
d) inducing an aerobic condition with ~aid micro~
organisms;
e) feeding said liquor containing nutrients into said
reactor and rnixing it therein with said microorganisms;
f~ allowing bio-degradation of said liquor by means
of said microorganisms and their multiplication thereby
1'` ~ '
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producing a biomass-containing body.
In other words, it can be stated that the liquor which
is used with nutrients is treated under non-aseptic conditions
with acclimatized microorganisms which are derived frorn sewage.
Although any liquor containing refuse fermentable
materials can be used, it is of course much more interesting, on
an industrial basis, to use one containing carboh~drates, more
particularly sulfite waste liquor which is thrown away in most
cases and can cause serious pollution problems. -
When the solid content of the liquor is too high, such
as when there i5 over 10% dissolved solids, the sulfite spent
waste liquor may be diluted with water~
For a more effective and practical operation of the
process accordin~ to the invention, it is preferable to use
acclimatized microorganisms. Ihis can be achieved by contact-
ing microorganisms from sewage with sulfite waste liquor for a
period of time varying between 10 and 20 days.
Although any nutrients known to those skilled in the
art may be added to the liquor before subjecting the latter
to bio-degradation with the preferably acclimatized micro-
organisms, it is preferred to use nitrogen-, phosphorus- and
potassium-containing nutrients. More preferably, the nutrients
supply nitrogen, phosphorus and potassium in a weight ratio
of about 12:2:1.
It is preferred to use amrnonia and KH2P0~ as the
source of nitrogen, phosphorus and potassiurn nutrients.
Of course, other nutrients, such as (NH4)2S04,
K2HP04 and the like well known to those skilled in the art
may also be used
Preferably, anti-foaming conditions should prevail
in the reactor, and these conditions may be obtained, for exam-
ple,by adding an anti-foaming agent such as the one manufactured
and sold by Dow Corning under the trade mark Antifoarn FG-10.
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It is recommended to have an aerobic condition in the
reactor and, for this purpose, for example, oxygen is conti-
nuously introduced into the fermentation rnix~ure in such a
way as to be continuously dissolved in the ranye between about
0.2 to 2~1 ppm oxygen. In general, the concentration of oxy-
gen in the mixture should be about 1~5 ppm.
For example, the oxygen may be introduced by adding
air to the reactor, either by resorting to mechanical means,
or by bubbling or the like, as it is well known to those
skilled in the art.
In order to provide an aerobic condition, it may be
easier to aerate a high capacity fermentation reactor than a
small reactor because of the longer residence time of the
air bubbles within the suspenslon.
As pointed out above, the ac7ldition of an anti foaming
agent or the use of anti-foaming conditions are helpful to
maintain steady-state operating conditic;ns.
In order to obtain good yields in biomass production,
it has been found that the pII in the xeactor should preferably
be adjusted to vary between about 4 and 7.
Adjusting the pH can be carried out by adding any
suitable agent. It has been found that an alkaline agent such
as sodium hydroxide, potassium hydroxide and ammonia can be
used, ammonia being preferred.
The microorganism3 consume the components oE the li-
quor in a preEerential manner, i.e. sorne nutrients which are
more difficult to dige~t are :Le~t aside. For example, the
Liynosulfonates are almost not bio-degraded by the mi~roorga-
nisms. In orcler to achieve the best possible results, the
residence of the liquor in the reactor has been found to be
preferably about 72 hours, As a matter of fact, it has
~.
been found that the microorganisms become acclimatized to the
sulfite waste liquor as a result of some competition between
the various microorganisms which are derived from domestic
sewage. For these reasons, under the conditions of the
reaction, development of predominant classe3 of microorganisms
will take place which has a definite influence on the nature
of the biomass which is obtained in final analysis.
Although a variation in temperature does not have a
harmful influence on the characteristics and properties of
the biomass which i5 obtained by the process according to the
invention, it has been found that in order to have the best
suitable conditions for producing good yields o biomass of
good quality the temperature should be adjusted to between
about 15 to 40~C, preferably between about 20 and 22~C,
otherwise the residence time of the liquor and the nature of
the microorganisms in the reactor will have to be substantially
adjusted. More preferably, the temperature in the reactor
should be maintained at about 20C.
In accordance with a preferred embodiment of the in-
vention, once a biomass-containing body is discharged from the
reactor, the biomass is dewatered, and this may be followed
by washing, sterilization, drying and grinding.
The microorganisms which are derived from domestic
sewage usually comprise a mixture of bacteria, yeasts and
mi.crofungi.
Broadly stated, the product obtained by the process
according to the invention conta.in~ when dried:
water 0.1 - 6 weight percent
proteins 25 - 50 "
minerals 0.1 - 5
vitarnins0.001 - .~ " "
-5-
.,, .... ..... , , ~,,. " .,,, ,. .. " ,.. .~ .. ~.......... .
r~
The invention will be illustrated by means of the
enclosed drawings, which are given only for the purpose of
illustration. In the drawings,
Figure l is a schematic representation of the system
used to produce dry biomass.
I~he in~ention will now be further illustrated by
means of the following example which should be read in con-
junction with the annexed drawings.
PRODUCTION OF SLUDGE SOLIDS ( BIOMASS )
Waste liquor
An industrial waste liquor from high-yield sodium
bisulfite pulping was used to produce the sludge solids. The
routine cooking conditions used in the pulping process are
shown in Table l.
TABLE I
ROUTINE COOKING CONDITIONS USED IN HIGH YIELD BIStJLFITE
PULP ING
_ _ . _
Wood species, wt. /O 94-93 softwood
and 6-7 hardwood
Fresh liquor/wood, w/w 4 : l
Total S02, wt. % 2.9
Cooking time at maximum temperature
(165C) 6 h
pH 4 - 5
Yield, Wt.% 70 - 75
_ _ _ . . . (pll,l,p)
The dry solids in this liquor normally contain about
50% sodium lignosulfonates and 40% carbohydrates expressed as
apparent glucose. The content of lignosulfonates was deter-
mined with W absorption at 280 nrn, and that of apparent glu-
cose by the anthrone reaction as described by Lo, S.N. and
r~ ~
Garceau, J.J., Can. J. Chem. Eng. 53,582 ~1975). 'I~is liquor
contains 6% to 10% dissolved solids and has a BOD5 approxi-
mately equal to 0.33 g 02/g dissolved solids.
Conversion of carbohydrates in liquor to biomass
In a preparation tank 1, waste liquor was first mixed
with nutrients, K2HP04 ancl (~4)2S04, to achieve a ratio of
BOD5 : N : P = 100 : 5 : 1 in the feed, if necessary, the
liquor was diluted with water in order to obtain 6% liquor so-
lids~ The pH in the feed was adjusted to 7 with ~aOH. The
fortified liquor was fed continuously via a feed tank 2 into
an aerated, cylindrical tank reactor 3 having a working volume
of 400 1, simultaneously, the reactor content was discharged
at a flow rate equal to that of the feed. The reactor contain-
ing acclimatized microorganisms originating from a muni-
cipal sewage treatment plant was operated under nonaseptic
conditions at pH's between 4 and 7. The
adjustment of pH to 7 was carried out manually, once per day,
with NaOH solution. Ihe foam was controlled with Dow Corning
antifoam FG-10* (food grade) dispersed in a large quantity
of water by means of an antifoam sprayer. During operations,
no difference was observed between the effluent and feed
temperatures, which were equal to room temperature maintained ~ -
at 20 - l~C~ Air, which also provided good mixing in the
reactor, was fed to -the reactor through a perfora-ted poly-
ethylene tube of 12.7 ~n I.D. Th.is tube, ln form of a coil
with several turns, was fixed on the reactor bottom. Dissolved
oxygen was measured with a Yellow Sprinc~3 Instrument (YSI)
oxygen meter, model 57. The reactox output contained approxi-
mately 11 y dry biomass/1. The recovery of this hiomass was
first carried out by sedimentation in settling tank 4, then by
batch-wise centrifugation at 5 which produced a paste con-
* Trade Mark
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taining 20 to 25% dry matter.
The sterilization and washing of this paste, which had
been placed in a cotton bag, was carried out at 6 simultaneous-
ly by holding the bag in boiling water for at least 30 min. The
volume ratio of boiling water to paste was greater than 10. Af-
ter allowing it to drip for a few hours the pagte was dried over-
night in a vacuum oven 7 at about 65C. The driecl product
was then ground into powder at 8. The product was beige and
had a very slightly salty taste and a very mild pleasant odour.
Table 2 shows the operating conditlons used in the
conversion of carbohydrates into a dry biomass of a mixed
culture from natural origin. In Table 2 it can be noticed that
a 6% liquor solids content and a 72-h hydraulic retention time
or sludge age were used in the operation.
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TABLE 2
OPERATING CONDI~IONS USED IN TME CONVERSION OF C~RBOHYDRArrES
TO DRY BIOMASS
_ _ ~
Solids content of waste liquor, wt. % 6
Density of waste liquor, kg/m 1026
Carbohydrates concentration in feed, g/l* 24.6
pH of feed 7
100 5 : 1
Reactor vol~ne, 1 400
Operating temperature, C 20
Hydraulic retention time, h 72
Mass concentration of microorganisms in the
reactor, g/l 10 to 11
Food to microorganisms, F/M, g carbohydrates
in feed/(g biomass in reactor) . d 0.78
Concentration of dissolved 2 in the reactor, ~ 1.5
pH in the reactor 4 to 7
Antifoaming agent, made of Dow Corning anti-
foam FG-10 and water, antifoam FG-10 :
water (v/v) 8 : 1000
Frequency of spraying antifoaming agent, min 1
~nount of antifoam agant sprayed in each time,
cm3 2.6
Total volume of the sedimentation unit, (two
equal units in series) 1 80
Detention time in each settling unit, h 7.2
Biomass content of the sediment in the first
settling unit, wt. % 1.8 to 2.2
Biornass content of the paste obtained after
centrifugation at 4,000 ~/min for 4 rnin, wt.~/~ 20 to 25
Washiny water : paste, ~/v 10 : 1
Time of sterilization at 100C, rnin 30
Drying temperature under approximately 100
kN m 2 vacuum, C 60 to 70
* expressed as apparent glucose.
g_
.
The microbial flora of the product according to the
invention consisted of a mixed culture of rnicxoorganisms,
originating from those present in the mixed liquor of -the
conventional domestic sewage -treatment process, i.e~ -the acti-
vated sludge process. Before putting the production system
into operation, sterilization experiments were run to determine
the time needed to kill in the produced biomass all the vegeta-
tive cells and spores, both aerobic and anaerobic. Experiment-
al results, obtained under aerobic and anaerobic conditions
revealed that boiling at 100C for 20 min. had killed both kinds
of microorganisms and the spores. In order to obtain a safer
final product the paste from the centrifugation step was then
sterilized by heating for 30 min. at 100C.
The overall composition of the product according to
the invention is presented in Table 3.
TABLE 3
OVERALL COMPOSITION OF MIXED-CULTURE MICROORGANISMS
Nitrogen-containing material or crude protein........ 37.9 wt. %
Moisture............................................. 4.4 wt. %
20 Ash................. ~................... ~.............. 4.1 wt. %
Fat............... . .................................. l wt. %
Carbohydrate, as apparent glucose 47.5 wt. %
Minerals_ mg/(kq dry matter)
. . _ .
Ca 1020
Fe _ 130
K 2750
. .
Mg 127.6
Na 11900
.. . .. . .. . .. ..... . .
P 6920
I~ere was found no trace of mercury or arsenic.
; The amino acid cont~nts of the product according to
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the invention are shown in Table 4.
TABLE 4
AMINO ACID COMPOSITION (q/16q NITROGEN)
Alanine 7~07
Arginine 4.72
Aspartic acid 8.04
Cy~tine
Glutamic acid 11.82
Glycine 4.48
Histidine 2,48 ~ -
Isoleucine 4.24
Leucine 8.73 -
Lysine 7.92
Methionine 3~06 -~
Norleucine 7.91
Phenylalanine 5.64
Proline 3.89
Serine 4.8
Threonine 3.72
Triptophan
Tyrosine 8.44
Valine 4-3
Some vi~amins have been measured. Their values are
presented in Table 5.
TABLE 5
; VITAMIN CO~TENTS OF MIXED CUETURE MICROORGANISMS
Vitamin m~/k~ drY matter
Thiamine.............. ,.......................... 5~66
Riboflavine........... ..................~........ 22.5
30 ~iacin.... ,..................................... llO
Folic acid......................................... 2.81
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t~
rrhe protein quality of the product according to the
invention was evaluated with young rats, using AOAC' 5 method
for PER determlnation. (Horwitz, W~, ed. "Official Methods of
Analysis", 12th Edi~ion (1975), Assoc. Official Anal. ~emists,
Washington, D.C. In the evaluation, ten youny male rats were
used in each group, thirty rats in total, the assay lasted
four weeks. In addition to using the product as the sole
source of protein, the diet of the assay yroup II was supple-
mented with 0.3 wt. % methionine. Table 6 gives the average
PER values obtained for the casein group and the two assay
groups. For comparison, these values were adjusted to an
assumed value of 2.5 for casein. Statistically, these values
are significantly different at a probability of less than
O ~ 01 .
These PER results show that the protein contained
in the product of the invention is short of sGme essential
amino acids, as indicated by the very low PER of 0~24 obtained
from assay group I. ~Iowever, when -the same protein was
slightly supplemented with methionine (0.3 wt.%), it gave a
PER of 2.05, close to that of casein for which the PER equals
2.5.
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TABLE 6
BIOLOGICAL EVALUATION OF PROTEIN 9UALITY:
.
PROTEIN-E~FICIENCY-RATIO*
-
ControlAssay Assay
~roup qroup I qrou~ II**
Average final weight, g 138.6 49.9 115.1
Average initial weight, g 45.7 45.9 44.~
Average weight gain, g 92.8 A4.0 B 70.3 A
Feed intake, g 285.5 A127~0 B271.8 A
Protein intake, g 30.3 A13.0 B28~0 A
Protein efficiency ratio, PER 3~06 A 0.3 B 2.51 C
Adjusted PER 2.5 A 0.24 B 2.05 C
_
* Ten rats in each group and four-week assay duration.
** The diet of this group was supplemented with 0.3 wt.%
methionine
tA, B, C) : Values in the same row with the same letter are
not significantly different at P ~ 0.01.
Tests made with chicken
During 4 weeks, young broiler chickens were fed with
two diets containing equivalent quantities of protein at maxi-
mum possible replacement levels derived from standard soya
flour and from biomass. The chickens were later slaughtered,
dressed and cooked. The weight gains of the chicken were
` statistically the same with both diets. Their carcass and
meat were found to be satisfactory upon veterinary inspection.
When cooked the meat of both types of chicken was found to
be equally satisfactory with respect to flavour and ~enderness.
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