Appl Microbiol Biotechnol (2006) 72: 480–485
DOI 10.1007/s00253-005-0297-y
BIOTECHNOLOG ICA L PROD UCTS A ND PRO CESS ENGINE ERIN G
E. Efremenko . O. Spiricheva .
S. Varfolomeyev . V. Lozinsky
Rhizopus oryzae fungus cells producing L(+)-lactic acid:
kinetic and metabolic parameters of free
and PVA-cryogel-entrapped mycelium
Received: 19 July 2005 / Revised: 7 December 2005 / Accepted: 9 December 2005 / Published online: 8 March 2006
# Springer-Verlag 2006
Abstract Spores of the filamentous fungus Rhizopus
oryzae were entrapped in macroporous poly(vinyl alcohol)
cryogel (PVA-cryogel). To prepare immobilised biocatalyst
capable of producing L(+)-lactic acid (LA), the fungus
cells were cultivated inside the carrier beads. The growth
parameters and metabolic activity of the suspended (free)
and immobilised cells producing LA in a batch process
were comparatively investigated. The immobilised cells
possessed increased resistance to high concentrations of
accumulated product and gave much higher yields of LA in
the iterative working cycle than the free cells did. Detailed
kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth,
substrate consumption and LA production showed that the
fungus cells entrapped in PVA-cryogel are more attractive
for biotechnological applications compared to the free
cells.
Introduction
A notable increase in the worldwide lactic acid (LA)
production is expected within a few coming years due to
the potential application of LA for the production of biodegradable polymers (Datta et al. 1995; Narayanan et al.
2004). The fungus Rhizopus oryzae is widely studied as a
E. Efremenko (*) . O. Spiricheva . S. Varfolomeyev
Chemical Enzymology Department, Chemistry Faculty,
The M.V. Lomonosov Moscow State University,
Lenin’s Hills, 1/11,
Moscow 119992, Russia
e-mail: [email protected]
Tel.: +7-95-9393170
Fax: +7-95-9395417
V. Lozinsky
A.N. Nesmeyanov Institute of Organoelement Compounds,
Russian Academy of Sciences,
Vavilov St. 28,
Moscow 119991, Russia
commercially perspective producer of L(+)-LA (Miura et
al. 2003; Yin et al. 1997), because the fungus cells possess
better resistance to high concentrations of accumulated LA
(Hamamci and Ryu, 1994; Schepers et al. 2002) compared
to the commonly used bacterial producers. Besides this
advantage, the fungi can use media with much lower contents of nutrient components compared to those required by
bacteria (Hujanen et al. 2001; Kwon et al. 2000). The use
of R. oryzae in immobilised form is one of the most
efficient approaches to improving the LA production process (Sun et al. 1999; Tay and Yang 2002; Xuemei et al.
1999). Immobilisation makes separating the liquid medium
from the cells much easier (Nedovic and Willaert 2004) and
facilitates multiple reuses of fungal cells for long-term LA
production.
The so-called poly(vinyl alcohol) cryogel (PVA-cryogel), a macroporous carrier prepared through the freeze–
thaw procedure (Lozinsky, 1998), is one of the most
promising carriers used for cell immobilisation (Lozinsky
and Plieva, 1998). The PVA-cryogel with high osmotic
stability and mechanical strength can be employed in
reactors with improved mass-transfer characteristics often
required for immobilised aerobic fungus cultures.
Preparing immobilised biocatalyst (IB) based on fungus
cells usually consists of the two main steps: the entrapment
of spores in the carrier matrix and further vegetation of
mycelium inside the granules (Dong et al. 1996; Xuemei et
al. 1999). Comparative analysis of process parameters obtained for suspended cells and immobilised cells is necessary to confirm the advantage of the latter and the
prospects of IB application in practice.
This work describes the study of the main kinetic and
metabolic parameters of the R. oryzae cells suspended and
entrapped in PVA-cryogel. The investigation of the most
important stages of IB formation is concurrently associated
with the growth of free cells under the same conditions.
Our preliminary studies have shown the prospects of
implementing the use of PVA cryogel in the preparation of
fungus-containing IB for LA production (Efremenko et al.
2004, 2005, 2006). Additional results are discussed in this
paper in more detail.
481
Materials and methods
Microorganism and immobilisation procedure
Fungus strain R. oryzae NRRL-395 was maintained and
grown for spore accumulation on a potato-dextrose agar
(Tay and Yang 2002). The liquid glucose-containing medium used for LA production was as follows (g l−1): glucose,
120; (NH4)2SO4, 3.0; MgSO4×7H2O, 0.3; ZnSO4×7H2O,
0.05; KH2PO4, 0.2 (pH 6.0).
The spherical beads of IB (1÷1.5 mm in diameter) were
prepared by entrapment of R. oryzae spores into PVAcryogel followed by the mycelium growth inside the carrier
matrix according to the patented procedure of Efremenko et
al. (2005). Immobilisation of the spores was accomplished
using a cryogranulating set-up (Lozinsky and Zubov
1992). The PVA (trade mark 16/1) utilised in the work
was purchased from National Producing Organization
(NPO) Azot (Severodonetsk, Ukraine).
Cultivation conditions
To investigate the kinetic and metabolic parameters of
fungus cell growth and LA production, the free spores
suspended in 0.9% NaCl or granules with immobilised
spores were inoculated into the culture medium so that the
initial concentrations of the cells were actually the same,
precisely (6.8±1.2)×107 spores ml−1.
Two batch cycles of cultivation of the suspended cells
and the immobilised cells were carried out on a shaker (Labtherm, Adolf Kühner, Switzerland) under aerobic conditions
(220 rpm, 28°C) in 500-ml Erlenmeyer flasks with 100 ml of
the medium. The duration of the first cycle was 120 h, and
the second one lasted 90 h. Both the free and the immobilised fungus cells were washed with 20 mM K/Na-phosphate
buffer (pH 6.8) between two batch cycles. To maintain pH
at the optimal level, CaCO3 (10 g l−1), preliminarily
sterilised in a dry form, was added to the nutrient medium.
Analytical methods
To analyse various parameters of cell growth and metabolic
activity, 66 flasks with free or immobilised cells were
concurrently used. Periodically, a flask triplet was taken out
of the process to determine the characteristics of the free
cells or the immobilised cells. Each registered value was an
average of three replicates. It was assumed that the amount
of PVA cryogel in each flask with IB was constant
throughout the cell cultivation process and equal to the
initial amount introduced with immobilised spores.
To determine the dry matter content in the suspended
cells and the immobilised cells, the samples were filtered
out using weighed de-ashed filters (Whatman No. 4), then
washed with sterile tap water and dried at 80°C until constant dry weights were registered. The dry matter amount of
the polymer carrier was deducted from the IB dry mass to
calculate the immobilised biomass.
The total LA concentration was measured by Econo
System (Bio-Rad, USA) high performance liquid chromatography using BioRad Aminex HPX-87H ion-exclusion
column (300×7.8 mm). The eluent, 0.005 M H2SO4, was
used at the flow rate 0.8 ml/min and the column temperature
80°Ñ. Concentrations of L-LA and D-glucose were assayed
by the enzymatic methods using the L(+)-lactate oxidase–
peroxidase kit (Sentinel, Italy) and the glucose oxidase–
peroxidase kit (Impact, Russia), respectively.
Scanning electron microscopy micrographs were obtained using a JEOL JSM-5300LV scanning electron microscope (Interactive Corporation, Japan).
Intracellular adenosine triphosphate (ATP) concentrations in the free cells and the immobilised cells were
determined by the bioluminescent method using the
luciferine–luciferase reagent (Moscow, Russia) (Dementieva
et al. 2001). To extract ATP from the free fungus cells, an
aliquot (0.1 ml) of cell suspension in culture medium was
centrifuged (Beckman J2-21 centrifuge, USA) at 5,000×g
for 10 min. Then the biomass precipitate was weighed and
treated with 1 ml of dimethylsulfoxide (DMSO) for 5 min.
ATP was extracted from the immobilised cells by treatment
of weighed granules (0.1÷0.3 g) with 1 ml of DMSO for
5 min. The cell extracts (50 μl) were added to the microcuvettes with aliquots of luciferase reagent (50 μl), and the
bioluminescence intensity was measured on a microluminometer 3,560 (New Horizons Diagnostics, USA). The
precise ATP concentrations in the tested samples were
calculated using the calibration plot obtained for ATP
standards.
Kinetic studies
The following kinetic parameters characterising the growth
and the metabolic activity of fungus cells were studied: the
specific rate of growth (μ, h−1), the specific rate of product
formation (qP, grams of LA produced by 1 g of cell dry
weight per hour), the specific rate of substrate consumption
(qS, grams of glucose utilised by 1 g of cell dry weight per
hour), the product yield per substrate used (YP/S, grams of
LA per gram of utilised substrate), the biomass yield per
substrate used (YC/S, grams of cells per gram of utilised
substrate), the rate of product formation (QP, grams of LA
in 1 l of culture medium per hour), the substrate conversion
(YP/So, percentage of accumulated LA per initially introduced substrate) and the product inhibition constant
(Kip, grams of LA in 1 l of culture medium). All parameters
were calculated following the known approach (Pirt 1975).
Results
LA production by the actively growing free cells
and immobilised cells
To compare the growth of the suspended cells and the
immobilised cells, the cultivation process was conventionally divided into the following three major stages: the
482
active growth phase of the suspended cells (the first period
of cultivation), the stationary phase (the second period) and
cell cultivation after replacement of the used medium with
a fresh one (the third period) (Fig. 1). The total duration of
the stages examined herein was 210 h. Glucose, LA and
intracellular ATP concentrations, as well as the amount of
accumulated biomass in the culture medium, were the main
parameters determined throughout the fermentation process.
The kinetic analysis of the growth and the metabolic
activity of the suspended cells and the immobilised cells
during the first 40 h of cultivation revealed that the immobilised cells possess a slightly lower specific rate of
growth compared to the free cells (Table 1).
The correlation between the increase in the intracellular
ATP concentration in the free cells and the immobilised
cells (Fig. 1) and germination of spores followed by biomass accumulation in the medium was shown at the first
cultivation stage. The metabolic parameter of the immobilised fungus cells, characterising the transformation of
glucose to LA (qp), was better than that of the free cells
(Table 1). Nevertheless, the accumulation of immobilised
biomass with higher productive potential was less than in
the case of free cells with lower specific productivity. As a
result, the total process productivity and the concentration
of accumulated product determined for the analysed period
were quite similar for both suspended and immobilised R.
oryzae cells.
In a separate investigation, the product inhibition constant (Kip) was detected for both free and immobilised cells
taken after 40 h of their cultivation. The found values were
equal to 144 and 181 g l−1 for both free and immobilised
cells, respectively. Independently on the investigated cell
form, the pH of the culture medium slightly decreased
(from 6.0 down to 5.8) towards the end of the exponential
phase of cell growth. Analogous investigation, carried out
in the absence of calcium carbonate (data not shown) when
pH value in the medium decreased from 6.0 down to 4.5
within 8 h, revealed much smaller values of Kip for the free
cells and the immobilised cells, at 60 and 78 g l−1,
respectively.
LA production in the presence of high product
concentration
After the 40th hour of cultivation, when the accumulated LA
concentration was close to 70 g/l in the flasks with the free
cells and the immobilised cells, the cells’ behaviour, as well
as their kinetic and metabolic parameters, notably changed.
Determination of the specific intracellular ATP concentrations in the suspended and PVA-cryogel-entrapped fungus
cells showed the obvious decrease in their energetic status at
the second stage compared to first stage (Fig. 1a), whereas
the energetic resource of the immobilised fungus cells was
notably higher compared to the free cells (Fig. 1b).
The growth of the free fungus cells rather decreased, and
the stationary phase of cell growth (μ=0) was observed after
the 10th hour of the second cultivation period (Fig. 1a). A
fivefold decrease in substrate consumption by free fungus
cells was also observed, while the cells converted the total
amount of utilised glucose into LA. The sevenfold decrease
in the average process productivity was established during the
second period of free cell cultivation, and not the first one.
Conversely, the immobilised cells continued to grow
even with a lower specific growth rate compared to the first
cultivation period (Fig. 1b). The process productivity of the
immobilised mycelium was higher at this stage of cell
cultivation compared to that ensured by free cells. The
immobilised cells easily retained their metabolic activity at
104 g/l LA in the medium, and the substrate conversion
reached 87% (Table 1). Thus, a high concentration of the
main product and its yield, established herein for PVAcryogel-entrapped fungi, was comparable to the maximal
values known from the literature (Tay and Yang 2002).
Cell growth and LA production in the course
of iterative cultivation
Fig. 1 a, b Cell growth (▪), substrate consumption (•), product
accumulation (▴), change of total (∘) and specific intracellular ATP
concentrations (ρ) during the batch cultivation of suspended (a) and
immobilised (b) R. oryzae fungus cells. Replacement of the spent
medium with a fresh one is marked with an arrow
The transfer of fungus mycelium to a fresh medium was
performed to supply the cells with new nutrient sources and
to eliminate the negative influence of the metabolites on the
483
Table 1 Parameters characterising growth and substrate conversion showed by free and PVA-cryogel-entrapped R. oryzae cells
Sample
μ, h−1
qS, g glucose
utilised g−1
cells h−1
qP , g lactic
acid produced g−1
cells h−1
First period of cell cultivation (from the beginning up to 40 h)
Free cells
0.093±0.014
1.06±0.18
0.91±0.22
Immobilised cells 0.079±0.018
1.11±0.15
1.03±0.23
Second period of cell cultivation (from 40 up to 120 h)
Free cells
0
0.024±0.008
0.024±0.004
Immobilised cells 0.009±0.002
0.080±0.034
0.070±0.032
Third period of cell cultivation (from 120 up to 210 h)
Free cells
0
0.021±0.003
0.021±0.004
Immobilised cells 0.003±0.001
0.13±0.02
0.11±0.01
YC/S, g
cells g−1
substrate
utilised
YP/S, g
lactic
acid g−1
glucose
utilised
QP, g
lactic acid
l−1 h−1
Lactic acid,
g l−1
YP/S0,
%
0.09±0.01
0.07±0.02
0.91±0.01
0.93±0.02
2.20±0.38
1.95±0.35
74±3
72±4
61±3
60±3
0
0.12±0.02
0.96±0.01
0.88±0.02
0.32±0.08
0.54±0.15
86±3
104±6
72±3
87±5
0
0.03±0.01
1.00±0.01
0.97±0.02
0.31±0.05
1.04±0.18
15±2
98±4
13±2
82±3
Each value is an average of three replicates
cell metabolic characteristics and the parameters of the LA
production process.
The difference between the parameters determined for
the free cells and the immobilised cells was more profound
after the replacement of the used medium with a fresh one
(Fig. 1). The suspended cells were characterised by low
substrate conversion and LA productivity for the first 20 h
after the medium replacement, and then dramatic cell lysis
was observed.
Fig. 2 a–d Scanning electron
microscopy micrographs of the
surfaces of an empty PVA
cryogel granule (a, b) and that
with fungus cells (c, d), where
carrier (1) and fungus mycelium
(2) are marked with arrows
Conversely, the slow growth of immobilised cells was
revealed concurrently with a high productivity of PVAcryogel-entrapped fungus mycelium in the fresh medium.
A high rate of LA production was found during the third
cultivation period. The IB was able to ensure 82% substrate
conversion whereas the free cells effected only 15%
glucose transformation. The analysis of LA purity showed
that both free and immobilised cells produced 98.5±1.2%
of L(+)LA-isomer.
484
Discussion
The difference between the kinetic and metabolic parameters of the suspended cells and those entrapped in PVAcryogel was established in this work. The dissimilarity
became more and more pronounced throughout the cell
cultivation. Other authors (Junter et al. 2002; Kosakai et al.
1997), comparatively analysing growth parameters or cell
productivity of various free and immobilised microorganisms (fungi, bacteria and yeast), explained the existing
differences between the characteristics of the suspended
cells and the immobilised cells by the mass-transfer
limitations or by dissimilar morphology of the free cells
and the immobilised cells.
In our case, scanning electron microscopy studies of
both the empty PVA-cryogel granules (Fig. 2a,b) and the
granules with immobilised mycelium (Fig. 2c,d) demonstrated high matrix porosity preventing diffusion hindrance
problems on the one hand, and enabling the development
of active mycelium on the other. The following comment
should be made as to the morphology of the free cells and
the immobilised cells: The surfaces of the free cell pellets
and the granules covered with immobilised mycelium
taken at the end of the second period of cultivation looked
identical (data not shown). However, the wetnesses of the
samples of the free cells and the immobilised cells were
72±3% and 83±2%, respectively. Probably, the growth of
the free cells resulted in the extrusion of water from the
inner parts of the pellets formed by densely packed
mycelium, whereas the presence of a mechanically stable
macroporous carrier matrix determined the formation of an
IB granule structure with lower internal cell density and
higher accessibility to substrate and oxygen supply, as well
as higher product removal.
It is known that the presence of a solid carrier in the
cultivation medium usually results in the adhesion of
mycelium on its surface (Kosakai et al. 1997). The growth
of filamentous fungi in contact with carrier parts
guarantees: (a) the formation of fungus morphology with
high cell productivity, and (b) a good distribution of fungus
cells in the reactor. Probably, the case we had of PVAcryogel-entrapped R. oryzae cells is a similar situation.
The presence of calcium carbonate in the medium had
positive effects on the LA productivity of both free and
PVA-cryogel-entrapped cells. However, the latter cells
always showed higher resistance to LA concentrations
accumulated in the medium even without pH adjustment.
This feature of IB is very attractive for biotechnology,
because the product isolation from the medium with high
LA concentrations has technological advantages and can
therefore have economic benefits.
In conclusion, the growth kinetics and the metabolic
parameters peculiar to the R. oryzae fungus cells, both free
and PVA-cryogel-entrapped cells, in the process of LA
production were comparatively analysed in detail for the
first time. Good repeatability of the characteristics of the
immobilised cells obtained in this work and the wellreproducible high productive potential of the IB were
confirmed in a separate investigation (Efremenko et al.
2006) when immobilised fungus cells were multiply used
for LA production in the batch and semi-batch regimes.
It was shown that the macroporous structure of PVAcryogel used as carrier allows the immobilised cells to
actively grow and efficiently produce LA. The behaviour
of the immobilised fungus cells positively differed from
that of the free cells at all investigated stages. The
performed studies demonstrated that iterative use of
PVA-cryogel-entrapped fungus for biotechnological production of LA is very promising for practical applications.
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