Download Sugar beet syrups in lactic acid fermentation – Part I

Technology/Technologie
Timo Johannes Koch, Joachim Venus and Martin Bruhns
Sugar beet syrups in lactic acid fermentation – Part I
Zuckerrübensirupe für die Milchsäuregärung – Teil I
Biotechnological production of lactic acid has been studied
in various ways, e.g. microorganisms, fermentation processes, down-stream processes, fermentation substrates,
and fermentation nutrients. The problems for all processes
still are high costs for feedstock and fermentation nutrients.
The objective of this study is a general evaluation of sugar
beet thick juice from Pfeifer & Langen GmbH & Co. KG, Germany as a substrate for lactic acid production.
In a series of fermentation experiments the results based
on thick juice were comparable to those obtained using cane
raw sugar and even better than using conventional corn
starch as a fermentation subtrate. The most important findings for a later technical application are the high volumetric
productivity (up to 5.5 g · L–1 · h–1), and the optical purity of
the lactic acid (>99% ee l-LA).
Die biotechnologische Herstellung von Milchsäure wurde
bereits aus verschiedensten Gesichtspunkten untersucht,
z.B. seitens der verwendeten Mikroorganismen und Fermentationsverfahren inclusive Produktaufarbeitung sowie hinsichtlich der eingesetzten Substrate und Nährstoffe. Wie für
andere Bioprozesse spielen auch hier die Rohstoffkosten eine
entscheidende Rolle. Insofern bestand das Anliegen dieser
Untersuchungen darin, die generelle Eignung von Zuckerrübendicksaft (Pfeifer & Langen GmbH & Co. KG, Germany) als
Substrat für die Milchsäurefermentation zu bewerten.
In einer Reihe von Fermentationsversuchen konnten vergleichbare Ergebnisse wie mit parallel untersuchtem Rohrzucker,
aber deutlich bessere im Vergleich zu Maisstärke erzielt werden. Die wichtigsten Erkenntnisse hinsichtlich einer industriellen Umsetzung bestanden in der für Batch-Prozesse hohen
volumetrischen Produktivität (bis zu 5,5 g · L–1 · h–1) und optischen Reinheit der erzeugten l(+)-Milchsäure (>99% ee).
Key words: lactic acid, fermentation, thick juice
Schlagwörter: Milchsäure, Gärung, Dicksaft
1Introduction
Nowadays, biotechnological processes and bio-based products
are the focus of various studies as promising alternatives to
petrochemical routes and products. “White Biotechnology”
points to an emerging field in biotechnology with immense
potential due to utilization of biocatalysts for the production
of industrial scale products. Lactic acid (LA) is a bio-based
organic intermediate of high interest. In chemical industry it
serves as a building block for a variety of key chemicals or bulk
polymers, e.g. acrylic acid and poly (lactic acid) (PLA). Today,
lactic acid is widely used in the food, cosmetic, pharmaceutical, and chemical industry but has received increasing attention as a monomer for the production of PLA, a bio-based and
potentially biodegradable polymer [1, 2] On the assumption
of a worldwide lactic acid production of 350,000 t, global
lactic acid consumption is estimated to increase significantly
at a rate of about 12–15% per year [3]. Growth of demand for
lactic acid and its salts and esters in industrial applications will
be driven mainly by lactic acid-based polymers and, to a lower
degree, lactate solvents [4].
Worldwide research is advancing focused on the use of renewable raw materials as carbon substrates as well as nutrient
additive sources. In this context, there is a strong interest to
reduce costs for raw materials and to use renewable resources
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[5]. Therefore, feedstock aspects for bioprocesses, the utilization of residues, waste materials [6–10] and agricultural byproducts [11–14] come into the focus of public attention. A
promising option is to ferment by-products and intermediates
of the sugar production, e.g. raw/thick juice, which is cheaper
than pure sucrose. After dilution to the desired initial sugar
concentration and addition of nutrients, the juice can be fermented to lactic acid and/or lactate [15].
In the present work, the objective was to test the suitability of thick juice (from sugar beet), cane raw sugar and corn
starch as feedstocks for lactic acid fermentation. There are
some examples available for the use of thick juice in fermentation processes, mainly for bioethanol production [16, 17], but
there are no investigations published for the application of
thick juice in lactic acid fermentation so far.
2
Lactic acid fermentation of carbohydrates
2.1Substrates
Three different feedstocks, cane raw sugar from Réunion, thick
juice from Pfeifer & Langen GmbH & Co. KG, Germany (P&L)
and corn starch from Cargill were tested as substrates for lactic
acid fermentation.
In focus of these studies is the sugar beet thick juice of P&L
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which is an intermediate of the processing of sugar beet into
crystalline sugar. It is obtained by concentrating purified juice
extracted from sugar beet to a sucrose content of ~64–66%.
The scope of these studies was not only to evaluate the general
applicability in fermentation but also to have a first look at
potential benefits for nutrient substitution. Hence all substrates were analyzed for their amino acid content by HPLC
analysis. Further information on the substrate compositions
are listed in the Supplementary Information (Table 6). Dry
substance content (wDS), sucrose content (wS) and purity of the
substrates are listed in Table 1.
2.2 Fermentation of different carbohydrate sources
2.2.1 Corn starch
At first results are shown for the fermentation of corn starch.
Here this substrate acts as basic medium because it is not
introduced into fermentation directly but only after saccharification. The following graphs show the results of starch fermentation after enzymatic hydrolysis.
Figures 1 and 2 show the typical time course for product formation and substrate consumption. Lactic acid bacteria are
Table 1: Characterization of fermentation substrates
Cane raw sugar
Thick juice
Substrate code
SB-001
SB-002/005/006
99.88
68.24
wDS in %
98.43
64.14
wS in %
Purity in %
98.55
93.99
Corn starch
SB-003
89.07
generally able to degrade starch. However, in all experiments
the microorganisms were not capable of converting more than
60% of substrate into lactic acid.
2.2.2 Cane raw sugar
The second substrate tested was cane raw sugar. In contrast to
starch, cane sugar consists of sucrose as carbohydrate source.
Figure 3 and Figure 4 show the time course of lactic acid formation and sugar consumption, respectively. In all fermentation experiments nearly the total amount of available carbohydrates was converted into product.
Fig. 3: Time function of lactate concentration in two parallel batch
cultivations for cane raw sugar
Fig. 1: Time function of lactate concentration in two parallel batch
cultivations for corn starch
Fig. 2: Time function of glucose concentration in two parallel batch
cultivations for corn starch
Fig. 4: Time function of sucrose concentration in two parallel batch
cultivations for cane raw sugar
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2.2.3 Thick juice
The third substrate evaluated was thick juice from P&L. As in
the previous experiments using cane raw sugar, the carbohydrates are nearly quantitatively converted into product. The
slopes of the curves are comparable to the results obtained
from the cane sugar experiments. The results are shown in
Figures 5 and 6.
2.2.4 Comparing all three substrates at a glance
Although the cells were adapted to the different carbohydrates
during the pre-cultivation the product formation started after
a certain lag-phase between 4 to up to 8 h. The fermentations
were terminated after 48 h with comparable results for both,
the thick juice and the cane sugar. For the starch-based broth
there was only 60% of the final lactate concentration achieved
because the residual sugars could not be used completely by
the bacteria.
Figures 7 and 8 confirm the results of the previous investigations in general. Again, the two substrates thick juice and cane
sugar showed the best performance with respect to the product formation. There is a certain delay for the thick juice but
reaching the same lactate concentration after 30 h.
The comparison of some characteristic performance data
(Table 2) does not strictly indicate the optimum combination
of all selected parameters for one feedstock. The compromise
Fig. 7: Product formation using different carbon sources starting from a
shared pre-culture
Fig. 8: Productivity curve for the different carbon sources starting from a
shared pre-culture
Table 2: Performance results of all three substrates in fermentations
Parameter*
Cane raw
Thick juice
Corn starch
sugar
Substrate code
SB-001
SB-002
SB-004
Sugar consumed in %
98
89
54
Yield in %
81
84
86
5.61
4.08
2.18
Ø Productivity in g L–1 h–1
* Average values over all experiments
Fig. 5: Time function of lactate concentration in two parallel batch
cultivations for thick juice
leads to the thick juice as preferable substrate due to its price
which is expected to be lower than starch or cane raw sugar
in terms of carbohydrate content. The low conversion rate for
hydrolyzed starch cannot be explained by the carbohydrate
itself, as also the sucrose from the other substrates has to be
hydrolyzed to glucose and fructose by enzymes from the lactic
acid bacteria. Because all other reaction parameters were kept
constant reasons for the low utilisation of corn starch must
be insufficient availability of amino acids (see Supplementary
Information), inhibition by impurities or implemented by the
enzymes that inhibited the microorganisms. However, the
fermentation of thick juice resulted in a high lactate concentration by giving high yields.
2.3
Fig. 6: Time function of sucrose concentration in two parallel batch
cultivations for thick juice
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Influence of different sugar beet thick juices
Finally, the influence of variations in quality, properties, and
behaviour of the residual material thick juice was investigated.
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Table 3: Results of repeating fermentation experiments with different
thick juice sample
Parameter/Exp.No
Substrate Code
Total turnover in %
Yield in %
Max. productivity in g L–1 h–1
15
SB-006
93
77
4.90
14
SB-002
91
74
5.50
16
SB-005
86
74
4.93
For that purpose three different charges of thick juice were
evaluated in the same way based on a common pre-culture as
in the previous experiments.
Comparison of the different thick juice samples shows slight
differences (Table 3) between the samples. One the one hand
this was expected due to the seasonal/regional character and
composition of the sugar beet raw material. One the other
hand, comparing the results of Tables 2 and 3 (substrate code
SB-002), the observed variation between several thick juice
samples are on a negligible scale which can be explained by the
expected variation between the individual fermentation runs.
Figures 9 and 10 show the time dependent concentration
profile of the substrate and the product during fermentation.
Both plots point out that all three experiments have a high
correlation over the complete reaction time.
2.4
With respect to the further processing of lactic acid to PLA
there is a need to guarantee high quality of the fermentation
product not only
according to the
Table 4: Illustration of the enantiopurity
impurities caused
(enantiomeric excess ee of l(+)-lactic acid)*
by the feedstock
Feedstock d(-) in g/L l(+) in g/L
ee / %
itself or by the
ThJ
0.4
95.34
99.16
nutr ient bro th
ThJ
0.22
95.46
99.54
but also in terms
CS
0.22
98.22
99.55
of the enantiopuCS
0.24
94.1
99.49
rity. For the presMSt
0
57.6
100
ent set of experiMSt
0
55.32
100
ments a strain
CS
0.5
97.52
98.98
was selected with
MSt
0.6
57.24
97.93
a stable capabilThJ
0.7
90.1
98.46
ThJ
0
83.98
100
ity of l(+)-lactate
ThJ
0.18
75.1
99.52
production. Table
ThJ
0.12
66.44
99.64
4 illustrates the
ThJ
0.24
88.88
99.46
enantioselectivity
ThJ
0.26
86.34
99.4
for all given indi* Additional data in Supplementary Information
vidual fermentaafter Acknowledgements.
tion r uns after
48 h with minimum enantiomeric excess of 97.93 independent of the raw
material used.
2.5
Fig. 9: Substrate concentration profile for three different thick juice
fermentations
Fig. 10: Lactate concentration profile for three different thick juice
fermentations
Enantioselectivity in fermentation of different
substrates
Potential of thick juice as nutrient
Microorganisms not only need a substrate to be converted to
a product but also various nutrients for their metabolism and
growth. Studying this question is quite sophisticated, as nutrient demand depends on many parameters. Indeed only fermentation experiments can give a true answer whether a substrate can also serve as nutrient source or not. A closer look at
the analytical data might also give a first hint. Most important
are the amino acids, not only in terms of the metabolism of
microorganisms but also in terms of the process costs. Especially for lactic acid bacteria several amino acids that differ
from strain to strain are essential. The experiments were carried out using relatively high amounts of an additional nutrient source (yeast extract). To reduce the costs of fermentation
media, the amount of added complex media components must
be low as possible.
The comparison of amino acids determined in corn starch,
crystalline sucrose (cane raw sugar) and thick juice shows two
facts (Table 6). First of all, thick juice contains a significantly
higher amount of amino acids than crystalline sucrose and
starch that hardly contain any of them. Second, there exists
a broad spectrum of amino acids in the juice. Hence these
data indicate that during fermentation thick juice might act
in a bifunctional manner, as substrate and as nutrient source.
Therefore, a reduction of the additional nutrient source seems
possible only with thick juice due its high content of amino
acids. This underlines the high potential of thick juice as a
fermentation substrate.
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3Conclusion
The study of alternative substrates for the production of lactic
acid to cut production costs is a major effort to implement an
industrial production. A first series of fermentation experiments with sugar beet thick juice, a sucrose-containing intermediate of beet sugar production, provided by P&L, shows that
this substance serves as a suitable substrate for the production
of lactic acid. In fermentation experiments the derived results
with thick juice were comparable to those obtained using cane
raw sugar and even better than using conventional corn starch
as a fermentation subtrate.
Beyond that, by comparing thick juices from different factories
and different beet campaigns it could be shown that different
thick juice qualities do not have a significant influence on the
fermentation performance.
Although not proven by fermentation experiments yet, analytical data show that thick juice might be co-utilized as a
nutrient source. Hence thick juice is not only technologically
more favourable and cheaper than crystalline sucrose, moreover it can lead to further cost saving by reducing the amount
of fermentation nutrients needed.
The experimental results of all substrates strongly hint towards
optimization of a process with thick juice as substrate. These
assumptions and more detailed questions regarding identification of suitable microorganisms, optimization of process
parameters, and continuation of the comparison of different
feedstocks have to be answered by further investigations.
4
4.1
Materials and methods
Bacterial strain and pre-culture preparation
According to previous experiments with thick juice as fermentation substrate the strain No. A35 (internal source) was
used. The pre-cultivation of the strain was carried out in shake
flasks overnight at 52 °C using the MRS bouillon No. 1.10661
(Merck, 64271 Darmstadt, Germany) as a medium while substituting the standard carbon source (glucose) by that one
which has to be tested in the subsequent lab-scale fermentation run. The volume of inoculum was set at 180 mL in each of
the experiments carried out.
4.2
ing Potsdam-Bornim e.V. (ATB) by P&L. The composition of
these compounds is described by the analytical data in the
Supplementary Information. The corn starch (C-Gel03402,
Lot: 03157184) is a product of Cargill, Neuss/Germany, the
cane raw sugar a product from an unknown source in Réunion
and the thick juice is a product of P&L. Yeast extract from
Deutsche Hefewerke GmbH, Germany No. 20901001 was
used as nutrient source. All other chemicals were technical
quality.
4.3 Fermentation experiments
4.3.1 Pre-treatment of the raw materials
For the preparation of the feedstocks it was necessary to break
down the corn starch to sugars to serve as a carbon source. The
enzymatic starch hydrolysis was carried out in three subsequent steps. After a short-term (30 min, 55 °C) pre-hydrolysis
of the insoluble starch by application of the enzymes BAN
480L in combination with Novozym50024 (close to the recommendations of Novozymes, DK) the liquefaction takes place at
higher temperatures (3 h, 80 °C) by means of an a-amylase
(Termamyl SC DS, Novozymes). Thirdly, the liquified starch
is degraded down to glucose by a glucoamylase (Dextrozyme
GA 1.5X, Novozymes) at moderate temperatures (3 h, 60 °C).
The mixture mainly containing glucose can be used as a basic
medium (2.5 L while adding the other nutrients up to 3 L
initial volume) in batch fermentation. The other two carbon sources (thick juice and sucrose/cane-sugar) were added
directly to the nutrient broth with an initial concentration
of 120 g/L carbohydrate mixed together with the nutrients
(yeast extract and salts, refer to Table 5).
4.3.2 Conditions of cultivation
Batch cultivations (3 L liquid volume) were carried out
in several lab scale stirred tank reactors (BIOSTAT® B/MD
[Fig. 12], B. Braun Biotech International GmbH,Germany and
BIOSTAT®B plus, Sartorius Stedim, Germany respectively
equipped with a digital control unit DCU) under temperature
and pH value control. Fermentation parameters and broth
composition are listed in Table 5.
Substrates, nutrients and supply chemicals
All three substrates, thick juice, cane raw sugar and corn starch
were provided to Leibniz-Institute for Agricultural Engineer-
Table 5: Conditions
Fermentation
Broth composition
Parameter
Value
Parameter
Value in g L–1
pH value
6.0
Yeast extract
15
2
Temperature in °C
52
K2HPO4
0.1
Base in % NaOH
20
MgSO4
0.05
Volume in
3
MnSO4
Time in h
48
Carbon source*
120
200
Stirrer speed in min–1
* Pol. sugar (cane sugar, thick juice), corn starch
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Fig. 11: BIOSTAT® Bplus (Sartorius BBI Systems GmbH, Germany)
equipped with a digital control unit DCU
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4.4
Analytical methods
Aliquots of the fermentation broth were taken every 2 h to
analyze lactate, sugars (glucose, fructose, and sucrose – e.g.
Fig. 12). The concentration of lactate and sugars was measured after dilution with HPLC (Dionex) using a Eurokat H
column (300 × 8 mm, Knauer, Germany) and detector RI-71
with a detection limit of 0.01 g L –1. The mobile phase was
0.01 N H2SO4 using an isocratic elution with a flow rate of
0.8 mL min–1.
Abbreviations
CS
Cane raw sugar
DS
Dry substance
DCU Digital control unit
ee
Enantiomeric excess
LA
Lactic acid
MSt
Corn starch
PolPolarisation
P&L
Pfeifer & Langen GmbH & Co. KG, Germany
ThJ
Thick Juice
PLA
Poly(lactic acid)
Notes and references
1
2
3
4
5
6
7
8
9
10 Tang, Y.; Bu, L.X.; He, J.; Jiang, J.X. (2013): l(+)-Lactic acid production
from furfural residues and corn kernels with treated yeast as nutrients.
European Food Research and Technology 236, 365–371
11 Thomsen, M.H. (2005): Complex media from processing of agricultural
crops for microbial fermentation. Applied Microbiology and Biotechnology 68, 598–606
12 John, R.P. (2009): Biotechnological Potentials of Cassava Bagasse. In: Biotechnology for Agro-Industrial Residues, 225–237
13 Alonso, J.L.; Dominguez, H.; Garrote, G.; Gonzalez-Munoz, M.J.; Gullon,
B.; Moure, A.; Santos, V.; Vila, C.; Yanez, R. (2011): Biorefinery processes
for the integral valorization of agroindustrial and forestal wastes. CytaJournal of Food 9, 282–289
14 Li, Z.; Lu, J.; Yang, Z.; Han, L.; Tan, T. (2012): Utilization of white rice bran
for production of l-lactic acid. Biomass and Bioenergy 39, 53–58
15 Visser, D.; Breugel, J. van; Bruijn, J.M. de; and A’Campo, P. (2007): Lactic
acid from concentrated raw sugar beet juice.
16 Nakagawa, M. (1992): Itaconic Acid Fermentation with Modeled Beet
Thick Juice and Molasses by Aspergillus-Terreus K26. Hakkokogaku Kaishi-Journal of the Society of Fermentation Technology 70, 451–456
17 Oda, Y.; Nakamura, K.; Shinomiya, N.; Ohba, K. (2010 ): Ethanol fermentation of sugar beet thick juice diluted with crude cheese whey by the
flex yeast Kluyveromyces marxianus KD-15. Biomass and Bioenergy 34,
1263–1266
Acknowledgements
The authors are grateful to the entire bioconversion group
at ATB for their experimental work, in particular Mr. Roland
Schneider for the operation of the lab-scale fermenters. Further thanks are due to Thomas Häßler from Pfeifer & Langen
for helpful discussions in order to prepare the manuscript.
Jim Jem, K.; Pol, J. van der; Vos, S. de (2010): Microbial Lactic Acid, Its
Polymer Poly(lactic acid), and Their Industrial Applications. Microbiology Monographs
Castillo Martinez, F.A.; Balciunas, E.M.; Salgado, J.M.; Domínguez González,
J.M.; Converti, A.; Pinheiro de Souza Oliveira, R. (2013): Lactic acid properties, applications and production: A review. Trends in Food Science &
Technology 30, 70–83
Corma, A.; Iborra, S.; Velty, A. (2007): ChemiSupplementary Information
cal Routes for the Transformation of Biomass
into Chemicals. Chem. Rev. (Washington,
DC, U.S.) 107, 2411–2502
Malveda, M.P.; Blagoev, M.; Kishi, A. (2006):
CEH Marketing Reasearch Report Lactic Acids, its Salts and Esters. SRI Consulting
Li, Y.; Cui, F. (2010): Microbial Lactic Acid
Production from Renewable Resources. Sustainable Biotechnology Sources of Renewable
Energy
Pintado, J.; Guyot, J.P.; Raimbault, M. (1999):
Lactic acid production from mussel processing wastes with an amylolytic bacterial strain.
Enzyme Microb. Technol. 24 (8/9), 590–598
Huang, L.P.; Jin, B.; and Lant, P. (2005): Direct
fermentation of potato starch wastewater to
lactic acid by Rhizopus oryzae and Rhizopus arrhizus. Bioprocess and Biosystems Engineering 27, 229–238
Bischoff, K.M.; Liu, S.; Hughes, S.R.; Rich, J.O.
(2010): Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile Bacillus coagulans. Biotechnology Letters 32, 823–828
Ouyang, J.; Ma, R.; Zheng, Z.; Cai, C.; Zhang,
M.; Jiang, T. (2013): Open fermentative production of l-lactic acid by Bacillus sp. strain
NL01 using lignocellulosic hydrolyzates as
low-cost raw material Bioresource TechnolFig. 12: Example figure for analysis of aliquots of the fermentation broth that were taken every 2 h
ogy 135, 475–480
to analyze lactate, sugars (glucose, fructose, and sucrose
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Table 6: Analytical data of substrates1
Cane raw sugar
Code
SB-001
SB-002
Analysis No.
PE 10-099-1
PE 10-099-2
Thick juice
SB-005
PE-10-136
SB-006
PE 09-252-2/
PE10-255-ZA-1
Corn starch
SB-004
PE 10-126-1
General
DS in %
99.88
68.24
67.34
67.2
88.49
98.43
64.14
62.5
63.23
–
Sucrose in °Z3
98.55
93.99
29.81
94.09
–
Purity in %4
9.76
9.59
–
pH
6.592
Ash [on DS] in %
0.22
2.06
1.22
0.14
Inorganic nonsugars in mg/kg DS
Sodium
19
1,764
1,229
1,300
51
Ammonium
3
62
41
20
3
Potassium
657
4,736
4,644
5,500
20
Magnesium
53
9
4
–
10
Calcium
145
49
159
100
52
Chloride
289
185
391
200
56
Nitrite
<10
67
39
–
<5
Nitrate
3
344
384
300
21
Phosphate
5
–
8
Sulfate
159
856
890
100
133
Amino acids in mg/kg DS
Alanine
3.8
138
199
362
15
Glycine
–
124
123
138
–
Valine
–
126
150
92
–
Leucine
–
207
220
166
–
allo-Isoleucine
–
–
–
–
–
Isoleucine
–
196
232
167
–
Threonine
–
87
105
48
–
Serine + GABA
–
718
626
590
–
Proline
–
101
94
60
–
Asparagine
35
225
231
96
–
Aspartic acid
27
477
487
599
–
Methionine
–
31
29
22
–
Glutamic acid
–
–
668
607
–
Phenylalanine
–
52
49
32
–
Glutamine
–
845
–
–
–
Lysine
–
38
46
29
–
Histidine
–
8.2
24
–
–
Tyrosine
–
342
415
263
–
Tryptophan
–
82
86
52
–
1
 Analysis performed in the analytical department of Pfeifer & Langen KG. 2 50% aqueous solution.
3
 SB-005 by HPLC; all other sample by polarimetry. 4 Sucrose to DS ratio.
Table 8: List of fermentation runs with the related feedstocks
Cane raw sugar
Thick juice
Code
SB-001
SB-002
SB-005
Analysis No.
PE 10-099-1 PE 10-099-2
PE-10-136
run No. (ATB internal)
1&2
×
3&4
×
6&7
8
×
9
10
×
14
×
15
16
×
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SB-006
PE 09-252-2
Corn starch
SB-004
PE 10-126-1
×
×
×
Table 7: Analytical data of nutrient1
Yeast extract
Analysis No.
PE 10-255-ZA-2
General
DS in %
94.92
–
Sucrose2
Glucose
–
Fructose
–
Purity3
pH
Ash [on DS]
Inorganic nonsugars in mg/kg
Sodium
2,200
Ammonium
9,050
Potassium
30,600
Magnesium
1,450
Calcium
1,650
Chloride
2,350
Nitrite
–
Nitrate
200
Phosphate
20,200
Sulfate
4,900
Amino acids in in mg/kg
Alanine
39.928
Glycine
15.757
Valine
34.885
Leucine
39.781
allo-Isoleucine
–
Isoleucine
28.003
Threonine
21.835
Serine + GABA
45.703
Proline
20.046
Asparagine
19.497
Aspartic acid
28.064
Methionine
9.022
Glutamic acid
57.451
Phenylalanine
22.404
Glutamine
–
Lysine
26.521
Histidine
8.390
Tyrosine
17.602
Tryptophan
5.989
1
 Analysis performed in the analytical department of Pfeifer & Langen KG. 2 SB-005 by HPLC
all other sample by polarimetry. 3 Sucrose to DS
ratio. Total N: 11.4%.
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Technology/Technologie
Table 9: Analytical data for enatiomeric analysis*
Feedstock run no. (ATB
d(-) in g/L
l(+) in g/L
HPLC in g/L
l/d [%]
l/d [ee]
internal)
ThJ
1
0.4
95.34
101.16
99.58
99.16
ThJ
2
0.22
95.46
100.25
99.77
99.54
CS
3
0.22
98.22
101.98
99.78
99.55
CS
4
0.24
94.1
100.13
99.75
99.49
MSt
6
0
57.6
60.54
100
100
MSt
7
0
55.32
59.42
100
100
CS
8
0.5
97.52
102.01
99.49
98.98
MSt
9
0.6
57.24
56.96
98.96
97.93
ThJ
10
0.7
90.1
93.45
99.23
98.46
ThJ
11
0
83.98
91.31
100
100
ThJ
12
0.18
75.1
80.35
99.76
99.52
ThJ
13
0.12
66.44
84
99.82
99.64
ThJ
14
0.24
88.88
93.54
99.73
99.46
ThJ
15
0.26
86.34
95.36
99.7
99.4
* In addition to the enantiopurity the accuracy of the analytical procedures has to be confirmed since
the correlation between the classical and chiral HPLC is sufficient. Only single values are out of the error
spread of 10%.
Authors’ addresses: Dr. Timo Johannes
Koch, Dr. Martin Bruhns, Pfeifer & Langen GmbH & Co. KG, Aachener Straße
1042 a, 50858 Köln, Germany; e-mail:
Timo.Johannes.Koch@Pfeifer-Langen.
com; Dr. Joachim Venus, Leibniz-Institute for Agricultural Engineering Potsdam-Bornim e.V. (ATB), Dept. Bioengineering, Max-Eyth-Allee 100, 14469
Potsdam, Germany; e-mail: [email protected]
Sugar Industry 139 (2014) No. 8 | 495–502