BIOUTILIZATION AND BIOTRANSFORMATION OF WASTES AND

Biotechnologia 3(1-2) 2004, 55-66
BIOUTILIZATION AND BIOTRANSFORMATION
OF WASTES AND BY-PRODUCTS
FROM FOOD INDUSTRY INTO ORGANIC ACIDS1
Waldemar Podgórski, ElŜbieta Gąsiorek, Władysław Leśniak,
Karol Gadomski
Wrocław University of Economics
Abstract. Cane and beet molasses processed via submerged methods gave citric acid
(CA) synthesis approximates to 100 g dm-3. Beet molasses can also be used for biochemical production of oxalic acid (OA) giving 38.7 g dm-3 of product. However, molasses is
not appropriate substrate for gluconic acid (GA) synthesis as only half of hydrolyzed saccharose can be converted by glucose oxidase to product. The same problems appear with
reference to sugar cane bagasse and sugar beet pulp, but these solid wastes can successfully be applied for other organic acid biosynthesis by solid-state method. Enrichment
them by carbon source allowed obtaining 132 and 204 g kg-1 d.s. of CA, respectively.
Hydrol bioconversion by submerged method gave high CA output approximates to
120 g dm-3. Mixture of wastes remaining after separation of gluten and starch from wheat
flour makes possible to obtain 30 g dm-3 of citric acid. Finally, the solid wastes, i.e. wastes coming from a filtration stage in glucose syrups purification, processed by solid state
fermentation, allowed obtaining 230 g of CA from approximately 1 kg of substrate used.
Key words: sugar and glucose production, by-products, wastes, Aspergillus niger, organic acid biosynthesis
INTRODUCTION
The control of environmental pollution and the recovery of agricultural, industrial,
and municipal wastes by converting them into useful products are nowadays the relevant biotechnological goals. From the other side the prerequisite for an economic yield
of organic acids bioproduction is the ability of microorganism to grow on a low cost of
carbon substrate. Thus using different wastes diminishing production cost makes the
process more attractive. The article deals with the application of organic wastes and by-
1
Corresponding author – Adres do korespondencji: Waldemar Podgórski, Department of Food
Biotechnology, Institute of Chemistry and Food Technology, Wroclaw Universitym of Economics, ul. Komandorska 118/120, 53-345 Wroclaw i e-mail: [email protected]
56
W. Podgórski i in.
products comes form white sugar production, corn processing into flour and flour processing into glucose and glucose syrups.
World sugar production for the 2003/04 marketing year is projected at 144.6 million
tons, raw value. Consumption is increased to 139.3 million tons [USDA 2003]. Approximately 75% of sugar is produced from sugar cane grown primarily in the tropical
and sub-tropical zones of the southern hemisphere with the balance produced from
sugar beet which is grown in the temperate zones of the northern hemisphere.
During sugar production, the following by-products and wastes are produced: molasses, sugar beet pulp, sugarcane bagasse, defecation mud, and spent wash. There are
many processes employing both cane and beet molasses as raw materials. Among them
alcohol production seems to be the most common. Nevertheless it is worth employing
such carbon sources for production more valuable substances. For example, the use of
sugar beet pulp to biosynthesis of organic acids increases range of application of this
by-product, which is usually used, as fodder for animals. This waste has never been
used as a substrate for e.g. citric acid production [Gąsiorek 1999].
In the process of potato, maize and wheat processing into glucose crystals and glucose syrups there also emerge some valuable wastes and by-products worth taking into
account as the potential raw materials for biotechnology. Among them are wastes from
production of starch syrups and crystalline monosaccharydes from wheat flour: hydrol the mother liquor remaining after the removal of crystals from concentrated dextrose syrup, wheat proteins (known as vital gluten), pentoses, less valuable starch granules and
filtration residues containing filtration residues and filtration bed material (cellulose).
Hydrol – is mainly used as a fodder additive and as a raw material for alcohol production. It was also examined as carbon and energy source for lysine [Rutkov et al.
1989] and citric acid biosynthesis [Leśniak et al. 1986, Wojtatowicz et al. 1991].
Wheat gluten is used to boost proteins level in food (e.g. in breakfast cereals, noodles and bakery and bread), as well as in fodder for animals. Mixture of wastes (MIX)
remaining after separation of gluten and starch from wheat flour consists mainly pentose sugars and less valuable starch grains. It is separated in a decanter as a light
fraction. These wastes are concentrated to approximately 20-30% of dry substance, and
in order to increase its suitability, wheat bran can be added. Obtained products are used
as very cheap fodder for animals. Filtration residues (FR) are so far not used.
The amount of by-products and wastes formed during starch syrups production is
equal approximately 25% of raw material (wheat flour) used. When, e.g., company
capacity requires 100,000 metric tons of wheat flour a year, the amount of by-products
formed will be as follows: 10,000 tons of gluten and 15,000 tons of other low economically valuable materials.
Taking into account above-mentioned wastes disposal, it is worth considering their
usage for manufacture of more valuable products. Thus the aim of this work is to show
the current state of investigations that have been made in our laboratory so far, pertaining to use of organic wastes and by-products comes form white sugar production, wheat
processing into flour and flour processing into glucose and glucose syrups to organic
acid biosynthesis.
Acta Sci. Pol.
Bioutilization and biotransformation...
57
MATERIALS AND METHODS
Materials
•
•
•
•
•
•
•
Bagasse – raw material of India origin.
Beet molasses – came from Polish sugar factory.
Cane molasses – from India.
FR – from Polish glucose syrups manufacturing factory. FR contained: sugars
35%, proteins 2%, cellulose fibers, and fats 63%. The sample used was characterized by: dry substance 64%, pH 4.4.
Hydrol – from Polish factory obtained by enzyme-enzyme procedure composed of water 48.9%, total sugars 45.6%, reducing sugars 39%, ash 1.03%,
total nitrogen 0.29%, and phosphorus as P2O5 0.15%. The pH was 4.0.
MIX from Polish glucose syrups manufacturing factory. MIX contained: water
74%, starch 28%, other sugars 20%, proteins 24%, fibers and fats 28% in dry
substance. The sample used was characterized by extract 18.65%, pH 4.0, reducing sugars 4.45%, and dry substance 26%.
Sugar beet pulp was obtained from sugar factory in Poland. It composed of
water 8-12%, dry substance 88-92%. Dry substance contained: cellulose and
hemicellulose 34-38%, pectins 34-40%, sugars 4-7%, proteins 6.5-8.0%, fats
0.5-0.7%, mineral salts 3.5-4.5 % [Nikiel 1978].
The liquid substrates with exception of MIX were diluted to approximately 120 g dm-3
of sugar with tap water then appropriate amounts of chemicals were added: NH4NO3,
(NH2)2CO (urea), H3PO4, KH2PO4, methanol, K4Fe(CN)6. The pH was adjusted to desired values using HCl solution.
Microorganism and cultivation conditions
Throughout the study the following Aspergillus niger strains were used: S, SBT,
C-12, W78B, W78C, and II. All strains belong to Department of Food Biotechnology
(Institute of Chemistry and Food Technology, Wroclaw University of Economics, Wroclaw, Poland) collection.
Biosynthesis methods
Solid-state fermentations (SSF):
Static solid-state fermentations – the 1000 cm3 glass beakers were filled with 50
grams of substrate (layer about 4.5 cm height). Substrate was moisturized to desired
humidity with tap water and sterilized (121°C, 30 min.). After cooling substrate was
inoculated with a suspension of Aspergillus niger spores in the amount of 1.8·106 spores/100 g. Fermentations were carried out in a thermostat at 30°C.
Dynamic solid-state fermentations were performed in horizontal rotating bioreactor Biosol (Food Biotechnology Department, Wroclaw University of Economics, Wroclaw, Poland) with the 7 dm3 total capacity (4.5 dm3 working volume).
Submerged fermentations (SmF):
Shaker - the 750-cm3 bulbs were filled with 125 cm3 of medium. The fermentations
were carried out at 26 - 29°C with the motion frequency of 180 min-1 and amplitude
6 cm.
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Laboratory bioreactors - fermentations were carried out in stirred tank reactors
(STR) MicroFerm Fermenter MF114 (New Brunswick Scientific Co. Inc., New Brunswick, New Jersey, USA), and Biomer 10 (Food Biotechnology Department, Wroclaw
University of Economics, Wroclaw, Poland) with the total capacity 14 and 7 dm3 respectively.
Analytical methods
The sample (about 4 grams) of fermented material from SSF was extracted during
24 hours with 50 cm3 of distilled water, and the extract was analyzed. The liquid sample
from SmF was examined after removing of biomass by vacuum filtration. During fermentation, biomass, sugar, total acidity, citric (CA), oxalic (OA), gluconic (GA) acids
were determined. Total acidity was analyzed by potentiometric titration method with
0,1 M NaOH to pH 9.0 or against phenolphthalein (5 cm3 sample from SSF, and 2 cm3
one from SmF).
Organic acids were assayed using isotachophoresis. Moreover, citric acid concentration was determined by pyridine and acetic anhydride method. CA was expressed as
monohydrate. Oxalic acid was precipitated in the filtrate of a sample after biomass removing with calcium chloride at boiling temperature. Then, the precipitate was dissolved in sulphuric acid, and the liberated oxalic acid was determined by titration with
potassium permanganate (KMnO4). OA was expressed as oxalic acid dihydrate.
RESULTS AND DISCUSSION
Optimal medium compositions for citric, gluconic and oxalic acids are simple (Table 1).
Precisely defined medium composition (pure sugar and source of N, Mg, P) allows
obtaining very high yield of product on total sugar (YP/S). In the case of citric and gluconic one, YP/S was higher then 90% (Table 3).
Table 1.
Optimal medium composition for organic biosynthesis in synthetic well-defined
medium
Tabela 1. Optymalny skład podłoŜa do biosyntezy kwasów organicznych
No
Medium constitution
Skład podłoŜa
Unit
J. m.
Value
Wartość
1
Carbon source (carbohydrates), źródło węgla
g dm-3
-3
120 - 200
2
NH4NO3
g dm
0.4 - 2.0
3
KH2PO4
g dm-3
0.2
-3
4
MgSO4·7H2O
g dm
0,2 - 0,9
5
(NH2)2CO (urea)
g dm-3
0.0 - 0.1
-3
6
Tap water*, woda wodociągowa*
dm
up to 1, do 1,0
7
pH
pH
2 -7
* Usually containing required number of microelements
* Zazwyczaj zawiera wystarczającą ilość mikroelementów
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Bioutilization and biotransformation...
59
The use naturals substrates as raw materials for organic acid biosynthesis causes difficulty coming from destruction of optimal medium composition. As a result, lower
both yield, and homofermentativity of the process are observed. The abundance of micro- and macroelements in such media are rather conducive to natural biomass growth
instead of pathological for Aspergillus niger citric acid overproduction.
Roots of sugar beet apart from sucrose (15-18%) and water, are composed of: pectins 1-2%, raw proteins 1.6%, fat 0.1%, fiber 1.0 -1.2%, N-free extract 12.6% and 0.81.0% of minerals [Morrison 1961, Ruffer and Rosenwinkel 1991]. Whereas, sugar cane
contains: proteins 1.0 %, fat 0.8%, fiber 6.8%, N-free extract 13.4% and 1.2% of minerals [Morrison 1961]. (Table 2). The extraction of sugar from beetroots or sugar cane
keeps the rest of plant composition in wastes abundant in various ingredients.
Protein
Białko
Fat
Tłuszcze
1
Sugar beet
Burak cukrowy
83.6
1.6
0.1
1.0
12.6
2
Beet molasses
Melasa buraczana
19.5
8.4
0
0
3
Sugar beet pulp
Wysłodki buraczane
82.0
10*
1.6*
4
Sugar cane
Trzcina cukrowa
76.8
1.0
26.6
8
5
6
Cane molasses
Melasa trzcinowa
Sugar cane bagasse
Bagassa trzcinowa
Mineral subst.
Substancje mineralne
Raw material
Surowiec
Fiber
Błonnik
No
Lp.
Water
Woda
N-free extract
Ekstrakt bezazotowy
Table 2. Average chemical composition of possible raw materials for organic acids productions [%]
Tabela 2. Skład surowców moŜliwych do zastosowania do produkcji kwasów organicznych [%]
Ca
P
N
K
1.1
0.04
0.04
0.26
0.25
62.0
10.0
0.05
0.02
1.34
4.77
61*
3.8*
5.1*
–
–
–
–
0.8
6.8
13.4
1.2
–
0.04
0.16
0.37
3.0
0
0
61.7
8.6
0.66
0.08
0.48
3.67
1.0*
–
49.0*
6.2*
–
0.90* 0.29*
–
0.5*
Source: [Morrison 1961] with exception of sugar beet pulp [Martinez and Gillman 1984] and sugarcane bagasse [Preston
2003], *Content in dry matter
Źródło: [Morrison 1961] z wyjątkiem wysłodków buraczanych [Martinez i Gillman 1984] oraz bagassy [Preston 2003],
*Zawartość w suchej masie
Therefore, in case of natural carbon and energy sources, medium composition hardly
can play a control role in microorganism’s metabolism. For example, using cane molasses for citric acid production, which is characterized as a changing and differentiating
substrate, the research experiments have suggested that the compiling of medium composition based on cane molasses is not sufficient for obtaining high efficiency of product formation. It turned out that the best results was possible to gain by the optimization
of culture aeration, allowed increasing product yield (YP/S) from 60 to 80% (Table 3).
Nevertheless, addition of specific substances (methanol, potassium ferrocyanide) to
prepared media, causing disturbances in natural metabolism of microbial producer,
appears to be also important.
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Table 3. Results of organic acids biosynthesis using different kind of raw materials
Tabela 3. Wyniki procesu biosyntezy kwasów organicznych przy zastosowaniu róŜnych
surowców
No
Lp.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Raw material
Surowiec
SM -White sugar
SM - Cukier biały
SM - Glucose
SM - Glukoza
SM - White sugar
SM - Cukier biały
Sugar cane bagasse
Bagassa trzcinowa
Sugar cane bagasse*
Bagassa trzcinowa *
Sugar beet pulp
Wysłodki buraczane
Sugar beet pulp
Wysłodki buraczane
Sugar beet pulp**
Wysłodki buraczane**
Sugar beet pulp
Wysłodki buraczane
Beet molasses
Melasa buraczana
Beet molasses
Melasa buraczana
Cane molasses
Melasa trzcinowa
Cane molasses
Melasa trzcinowa
MIX
MIX
Hydrol
Hydrol
FR-Filter Residuals
FR-Odpady filtracyjne
Main
product
Produkt
główny
Qp
SSF g kgg g-1 ·100 1h-1, SmF
g dm-3h-1
Main
product
Produkt
główny
YP/S
Method
of
fermentation
Metoda
fermentacji
Strain
Szczep
SmF-CA
W78B
150
139
0
0
92
0.92
SmF-GA
W78B
65
–
199
–
101
3.07
SmF-OA
W78C
144
43.8
26.5
79.8
40
0.42
SSF-CA
S
96
23
–
–
2.3
0.24
SSF-CA
S
96
132
–
–
13.2
1.38
SSF-CA
S
72
150
–
–
15.1
1.88
SSF-CA
S
120
173
–
–
17.3
1.44
SSF-CA
S
120
204
~ 88
20.4
2.13
SSF-GA
S
~72
28.3
93.3
1.33
9.3
1.30
SmF-CA
W78B
160
104
1.5
0.6
80
0.65
SmF-OA
II
144
~38
38.7
30
0.85
SmF-CA
W78B
160
100
2.4
1.6
80
0.63
SmF-GA
W78B
90
44
22.8
0.5
20.7
0.24
SmF-CA
S
200
–
–
64.7
0.14
SmF-CA
W78B
216
–
–
90.0
0.51
SSF-CA
S
168
–
–
22.8
1.36
t
CA
GA
OA
SSF - g kg-1,
SmF - g dm-3
h
* With 25% of tapioca, ** with addition of 20% of molasses.
* Z dodatkiem 25% tapioki, ** z dodatkiem 20% melasy.
E.g. SmF-CA means that the process was optimized in order to obtain the highest output of citric acid using submerged
fermentation.
Np.. SmF-CA oznacza, Ŝe proces był optymalizowany w celu maksymalizacji stęŜenia kwasu cytrynowego w fermentacji
metodą wgłębną.
All data were calculated as a mean value of at least two independent experiments.
Zamieszczone w tabeli dane są wartościami średnimi otrzymanymi z przynajmniej
dwu niezaleŜnych eksperymentów.
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Bioutilization and biotransformation...
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Summarizing, the control function of minimal medium composition (nutrient uptake
inhibition) for high product yield in case of by-products and wastes application has to
be replaced with other methods of metabolism control, i.e. strain selection and improvement, addition of chemical modifiers, dynamic methods of process control, etc.
[Podgórski 2002].
The fermentation techniques and methods of process control that we were about to
employ depended on raw material used. Thus, liquid sugar’s production wastes allowed
applying submerged (SmF) or surface method (SF) of biosynthesis, whereas for solid
one the solid-state method (SSF) was more appropriate. There are some other methods
of fermentations used by some research workers but they are employed mainly as a
compilation of above-mentioned basic methods. For example, Singh et al. [2003] analyzing production of gluconic acid with respect to varying substrate concentrations had
been used semi-solid state (SmSF) method. Nevertheless, the highest level of gluconic
acid 106.5 g dm-3 was obtained under SSF conditions. The maximum degree of gluconic
acid conversion was observed at initial substrate concentration of 120 g dm-3. SmF and
SmSF processes were completed within 6 days of incubation, whereas the highest yield
was observed after 12 days of incubation and continued thereafter in the SSF process.
In our experiments, much better results of GA synthesis were obtained using SmF
method (Table 3). The initial sugar concentration amounted to 200 g dm-3, and final
product formation was 199 g dm-3. The high results of the fermentation (rather biotransformation as only one enzyme glucose oxidase EC 1.1.3.4 in the process is involved)
were possible to obtain employing control parameters directly connected with metabolism of microorganism reflecting its currant activity in gluconic acid biosynthesis. The
relatively high correlation between values usually determined via sample analysis and
microorganisms’ respiratory activity enabled computer to on-line estimation of biomass
growth, product formation, and substrate uptake rate. These continuously measured
parameters employed to on-line control of gluconic acid overproduction allowed obtaining high effectiveness of product synthesis (YP/S) equals nearly 100% and product formation rate amounted to 5 g dm-3 h-1 [Podgórski 2004].
Bioutilization of liquid substances using SSF requires special carriers to achieve optimal fermentation space structure. The suitable material for this purpose turned out to
be the sugarcane bagasse. This matter alone is not valuable substrate, but in mixture
with other carbon source allows obtaining high product yield. For example Kumar et al.
[2003] using solid-state fermentation with sugar cane bagasse as a carrier and sucrose or
molasses based medium as a moistening agent obtained after 9 days of fermentation
20.2, and 19.8 g citric acid from 100 g-1 of dry solid with yield of 69.6, and 64.5% (based on sugar consumed) respectively.
The experiments with bagasse in our laboratory allowed obtaining more the 130 g
kg-1 of citric acid but productivity amounting to 1.38 g kg-1h-1 was higher than that obtained by Kumar and co-workers (Table 3), [Kumar et al. 2003]. Nevertheless, the highest productivity (2.13 g kg-1h-1) was achieved during SSF with sugar beet pulp and addition of 20% of molasses as carbon source (Table 3). More then 204 g of citric acid was
obtained during 120 hours of the process. No addition of molasses to the sugar beet pulp
also gave relatively high product formation amounted to 138 g kg-1h-1) (Table 4). The
optimal medium composition in last case was very simple, as only sugar beet pulp and
process water were necessary [Gąsiorek 1999].
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Table 4.
The influence of molasses addition to sugar beet pulp on biosynthesis of citric acid
in a horizontal rotating solid-state bioreactor
Tabela 4. Wpływ dodatku melasy do wysłodków buraczanych na biosyntezę kwasu cytrynowego
w poziomym, obrotowym reaktorze solid-state
No
Lp.
A
Molasses
dose [%]
Dodatek
melasy [%]
X
P
g kg-1
g kg-1
Days of fermentation
Dni fermentacji
1
2
3
4
5
Yield
in relation to
Wydajność
w stosunku do
dry subst.
sugar
suchej
cukru
substancji
%
%
Qp
g kg-1h-1
1
0
13.2
90.5
181.0 197.0 173.0
27.3
137.9
13.8
–
2
10
12.2
83.0
195.0 256.0 245.0
28.2
179.2
17.9
167.9
1.44
1.87
3
20
14.3
112.0
241.5 292.0 228.0
25.8
204.4
20.4
111.1
2.13
Source [Gąsiorek 1999]
Źródło [Gąsiorek 1999]
The solid state method did not allow using more then the 20 - 30% of liquid substrate in relation to solid carriers, thus the large amount of molasses has to be processed
by submerged method. This technique of fermentation assures good results with high
product yield. For example, Ikram-ul-Haq et al. [2002] carrying out citric acid fermentation using cane-molasses in 15 dm-3 stirred tank reactor obtained 106.65 g dm-3 of anhydrous citric acid. Our research (Table 3) gave 100 g of citric acid monohydrate with a
small amount of accompanying organic acids.
Molasses can also be used for biochemical production of oxalic acid [Podgórski and
Leśniak 2003] in spite of opinions that organic wastes including molasses are not suitable raw materials for OA production. Nevertheless, amount of product that was
possible to obtain was two-fold lower in relation to citric acid (Table 3). Molasses
however, seems not to be suitable carbon source for gluconic acid biosynthesis as only
half of hydrolyzed saccharose can be converted by glucose oxidase to product. In such
cases, fructose is metabolized to other organic acids. On the other hand, simultaneous
production of organic acids in one bioprocess seems to be also attractive.
Other valuable raw material for bioproducts formation is hydrol – the by-product
remaining after removal of crystals from concentrated dextrose syrup in a process of
glucose production. The amount of CA obtained in hydrol contained media was higher
then 120 g dm-3, giving 90% of product yield in relation to sugar consumed (YP/(S-SE) ).
Fermentations lasted 168 hours.
Apart from by-products formed during production of final products, there are also
numbers of wastes worth to be taken into account. Their bioutilization can lead to the
ecological, cleaner technologies becoming more friendly to the environment. We have
examined MIX - i.e. mixture of wastes remaining after separation of gluten and starch
from wheat flour processing, consisting mainly pentose sugars and less valuable starch
grains, and FR - wastes came from the filtration stage in glucose syrups purification contained filtration residues and filtration bed material (cellulose). Both these materials
were used as substrates for citric acid biosynthesis.
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The fermentation results with MIX substrate depicted in Table 5 show that in order
to obtain the yield of citric acid (YP/S) equals 64.7%, MIX waste has to be enriched by
0.2 g dm-3 of KH2PO4 source. The essential precondition is also an increase of initial pH
of the medium. The highest amount 28.8 g of product was obtained at pH equals 6.4.
The reason for relatively low product yields (YP/S) came from the small amount of easy
to uptake sugars content in the raw material (4.45%), and lack of assimilation other
medium ingredients (mainly pentoses).
Table 5.
Standard and optimum conditions for fermentation of MIX to produce of citric acid in
the SmF fermentation
Tabela 5. Warunki standardowe i optymalne produkcji kwasu cytrynowego przy zastosowaniu
odpadu MIX
Medium comp.
Skład podłoŜa
Substrate (S),
substrat (S)
Standard
value
Wartość
standardowa
Unit
J.m.
Interval Optimum
value
studied
Wartość
Zakres
zmian optymalna
t
P
YP/S
YP/S’
cm3 dm-3
1000
0.0
–
144
16.6
37.3
1.7
NH4NO3
g dm-3
1.0
0.0 - 2.0
0.0
144
15.9 - 16.6
37.3
1.6 - 1.7
KH2PO4
g dm-3
0.1
0.0 - 0.2
0.2
144
16.6 - 25.0
56.2
1.7 - 2.5
K4Fe(CN)6·3H2O
g dm-3
0.0
0.0 - 1.0
0.0 144-216
15.9 - 16.6
37.3
1.6 - 1.7
%
0.0
0.0 - 2.0
0.0 144-216
16.3 - 18.4
41.2
1.6 - 1.8
–
4.0
4.0 - 6.4
6.4 144-216
16.6 - 28.8
64.7
1.7 - 2.9
CH3OH
pH
All data calculated as a mean value of at least two independent experiments.
Zamieszczone w tabeli dane są wartościami średnimi otrzymanymi z przynajmniej dwu niezaleŜnych eksperymentów.
250
P(55) = - 0.0135t2 + 5.161t - 267.86
R2 = 0.9876
55
50
200
60
P [g kg -1]
150
65
100
70
50
60 (MIX)
0
24
48
72
96
120
144
168
192
216
t [h]
Fig. 1. The course of citric acid in static solid-state fermentation of FR wastes with different
humidity level. Numbers close to the curves denote initial humidity of the FR waste. The
MIX means that MIX was used as moistening agent
Rys. 1. Wpływ wilgotności odpadów FR na ilość syntezowanego kwasu cytrynowego. Liczby
obok krzywych oznaczają początkowe wilgotności odpadu FR. Przypis MIX oznacza, Ŝe
odpad ten był uŜyty jako czynnik nawilgacający odpad FR
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Much higher amount of product was obtained using FR waste as a carbon and energy source. In that case, citric acid formation was strongly affected by humidity of
substrate (Fig. 1). At the optimum moisture level equals 55%, maximum product formation amounted to 228 g dm-3 with YP/S = 65% and YP/S’ = 22.8% yields. Addition of
MIX waste as a moistening agent to the FR substrate caused worsening in process performance. It suggests that MIX may contain some unfavorable ingredients for high CA
biosynthesis.
CONCLUSIONS
Summarizing all experiments it is worth underlining, that application of appropriate
strains of Aspergillus niger and methods of fermentations allows using wide spectrum
of substrates, for biosynthesis of organic acids. They include by-products and wastes of
wheat processing and sugar production origin. Furthermore, it seems that resurgence of
solid-state method for cultivation of microorganism brings about a better utilization of
solid and semisolid wastes come from other agro-industrial production. It is especially
important nowadays as the economy and ecology of particular countries are strongly
dependent on utilization of this huge quantity of agro-industrial residues being produced. Among them are sugar cane bagasse, sugar beet pulp, apple pomace, coffee husk
and pulp, soybean defatted cake, declassified potatoes, wheat bran, etc. These wastes
apart from organic acids can be used for biotechnological production of many other
useful products such as protein, enzymes, food aroma compounds, biopesticides, mushrooms, pigments, xanthan gums, hormones (gibberellic acid), etc. [Soccol and Vandenberghe 2003].
LIST OF SYMBOLS AND ABBREVIATIONS
A
CA
P
QP
RS
S
SmF
SM
SmF
SmSF
SSF
t
X
YP/(S-SE)
– total acidity (as citric acid), g kg-1 – kwasowość ogólna g kg-1,
– citric acid – kwas cytrynowy,
– product SSF - g kg-1 d.s., SmF - g dm-3, produkt SSF – g kg-1 s.m. SmF –
g dm-3,
– productivity SSF - g kg-1 d.s. h-1, SF - g dm-3 h-1 – produktywność SSF –
g kg-1 s.m. h-1, SF - g dm-3 h-1,
– reducing sugars after hydrolysis – cukry redukujące po hydrolizie,
– concentration of reducing substances in medium (after hydrolysis),
SSF – g kg-1 d.s.,
– g dm-3 – stęŜenie substancji redukujących (po hydrolizie), SSF – g kg-1
s.m., SmF - g dm-3,
– synthetic medium – podłoŜe definiowane,
– submerged fermentation – fermentacja wgłębna,
– semisolid state fermentation – fermentacja w podłoŜu półstałym,
– solid-state fermentation – fermentacja w podłoŜu stałym,
– fermentation time, hours (h) – czas fermentacji, godziny (h),
– biomass concentration, g d.s. dm-3 – stęŜenie biomasy, g s.m. dm-3,
– yield factor of product on sugar consumed, 100 g g-1 – współczynnik wydajności substratowej w stosunku do cukrów wykorzystanych, 100 g g-1,
Acta Sci. Pol.
Bioutilization and biotransformation...
YP/S
YP/S’
65
– yield factor of product on total sugar, 100 g g-1 – współczynnik wydajności
substratowej w stosunku do cukrów ogółem, 100 g g-1,
– yield factor of product on substrate used, 100 g g-1 – współczynnik wydajności substratowej w stosunku do ilości uŜytego substratu, 100 g g-1.
REFERENCES
Gąsiorek E., 1999. The research on the biosynthesis of citric acid by solid state cultivation method. PhD thesis. Wroclaw. (In Polish).
Ikram-ul-Haq, Ali S, Qadeer M. A., Iqbal J., 2002. Citric acid fermentation by mutant strain of
Aspergillus niger GCMC-7 using molasses based medium. Electronic J. Biotechnol. 5 (2),
125-132.
Kumar D., Jain V. K., Shanker, G. Srivastava A., 2003. Citric acid production by solid state fermentation using sugarcane bagasse. Process Biochem. 38 (12), 1731-1738.
Leśniak W., Pietkiewicz J., Gąsiorek E., Podgórski W., 1999. The laboratory rotary bioreactor for
cultivation of micoorganisms in solid media - Biosol. 1st National Congress of Biotechnology. Postery. Wrocław 20-25.09.1999 r. p.76. (In Polish).
Leśniak W., Podgórski W. Pietkiewicz J., 1986. MoŜliwości zastosowania hydrolu glukozowego
do produkcji kwasu cytrynowego. Przem. Ferm. Owoc.-Warzyw. 30 (6), 22-25. (In Polish).
Martinez R., Gillman L., 1984. 2.3-Butanodiol production from sugar beet pulp. Third European
Congress on Biotechnology. II, 247-252.
Morrison F. B., 1961. Feeds and Feeding.. Abridged, 9th ed. The Morrison Publishing Co.
Nikiel S., 1978. Cukrownictwo. WsiP, Warszawa.
Podgórski W., 2002. Modeling of respiratory and acidogenesis activities of Aspergillus niger
during citric acid production in cane molasses media). Prace Nauk. 914. Monografie i opracowania nr 144. Wydawnictwo Akademii Ekonomicznej, Wrocław. (In Polish).
Podgórski W., 2004. Monitoring of A. niger metabolic activity in biotransformation of glucose to
gluconic acid. Biotechnologia. 1 (64), 169-186. (In Polish).
Podgórski W., Leśniak W., 2003. Biochemical method of oxalic acid production from beet molasses. Chem. Pap. 57 (6), 408-412. (In Polish).
Preston R. L., 2003. Feed com. The latest data on typical composition of commonly used feeds
for catle and sheep. Beef. www.beef-mag.com.
Ruffer H., Rosenwinkel K.H., 1991. Taschenbuch der Industrieabwasserreinigung. R. Oldenbourg
Verlag Munchen, Vien.
Rutkov A., Peikova S., Krusteva J., 1989. Glucose molasses hydrole as a substrate for microbial
production of l lysine. Acta Microbiol. Bul. 24, 69-75.
Singh O. V., Jain R. K., Singh, R. P., 2003. Gluconic acid production under varying fermentation
conditions by Aspergillus niger. J. Chem. Technol. Biotechnol. 78 (2-3), 208-212.
Soccol C. R., Vandenberghe L.P.S., 2003. Overview of applied solid-state fermentation in Brazil.
Biochem. Eng. J. 13 (2-3), 205-218.
USDA -The U.S. Department of Agriculture, 2003. USDA Foreign Agricultural Service. The
2003/04 world sugar production estimates. http://www.fas.usda.gov/psd/.
Wojtatowicz M., Rymowicz W., Kautola H., 1991. Comparison of different strains of the yeast
Yarrowia-lipolytica for citric-acid production from glucose hydrol. Appl. Biochem. Biotech.
31 (2), 165-174.
Biotechnologia 3(1-2) 2004
66
W. Podgórski i in.
BIOUTYLIZACJA I BIOTRANSFORMACJA
PRODUKTÓW UBOCZNYCH I ODPADÓW
PRZEMYSŁU SPOśYWCZEGO DO KWASÓW ORGANICZNYCH
Streszczenie. Melasy trzcinowe i buraczane przetwarzane metodą fermentacji wgłębnej
pozwalają na uzyskanie kwasu cytrynowego w ilości 100 g dm-3. Z mniejszą efektywnością (38,7 g dm-3) melasa buraczana moŜe być wykorzystana do produkcji kwasu szczawiowego. Surowiec ten nie nadaje się natomiast do produkcji kwasu glukonowego, jako
Ŝe tylko połowa cukrów w niej zawartych moŜe ulec biotransformacji do końcowego produktu. DuŜą przydatność do procesów biosyntezy wykazały bagassa trzciny cukrowej i
wysłodki buraczane pozwalając na uzyskanie odpowiednio ponad 130 i 200 g produktu z
1 kg substratu. Do produkcji kwasów organicznych moŜliwe jest takŜe zastosowanie odpadów powstających w procesie otrzymywania glukozy. UŜycie hydrolu pozwala na uzyskanie kwasu cytrynowego w ilości 120 g dm-3. Gorsze wyniki (30 g dm-3) otrzymano
przy zastosowaniu odpadów powstających w procesie separacji glutenu i skrobi. Wysoką
efektywnością fermentacji charakteryzuje się natomiast zastosowanie odpadów stałych
powstających w procesie filtracji syropów glukozowych. Ilość uzyskanego metodą solid-state kwasu cytrynowego z zastosowaniem tego odpadu wyniosła 230 g na 1 kg wykorzystanego surowca.
Słowa kluczowe: produkcja cukru i glukozy, produkty uboczne, odpady, bioutylizacja,
kwasy organiczne, Aspergillus niger
Accepted for print – Zaakceptowano do druku: 10.12.2004
Acta Sci. Pol.