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Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Effect of ammonium-N on malic enzyme andlipid production in Rhodotorula glutinis grown onmonosodium glutamate wastewater
Guiping Gong, Guiying Guo, Xu Zhang & Tianwei Tan
To cite this article: Guiping Gong, Guiying Guo, Xu Zhang & Tianwei Tan (2016) Effect ofammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown onmonosodium glutamate wastewater, Biocatalysis and Biotransformation, 34:1, 18-23, DOI:10.1080/10242422.2016.1201077
To link to this article: http://dx.doi.org/10.1080/10242422.2016.1201077
Published online: 16 Jul 2016.
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Biocatalysis and Biotransformation, 2016; 34(1):18–23
RESEARCH ARTICLE
Effect of ammonium-N on malic enzyme and lipid production in
Rhodotorula glutinis grown on monosodium glutamate wastewater
GUIPING GONG, GUIYING GUO, XU ZHANG & TIANWEI TAN
National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University ofChemical Technology, Beijing, China
Abstract
Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid
production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000 mg/L, COD 30,000–
50,000 mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial
ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140 g/L. With an increase in the
ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass
decreased at 72 h. The maximum value of ammonia nitrogen consumption reached 5.77 g/L for an initial ammonium sulfate
concentration of 140 g/L at 72 h. In addition, 60 g/L ammonium sulfate concentration may be an appropriate concentration
for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear
relationship between the intracellular lipid content and the total malic enzyme activity.
Keywords: Malic enzyme; microbial lipid; monosodium glutamate wastewater; Rhodotorula glutinis
Introduction
With declining reserves of fossil fuel and increasing
consumption of energy, renewable energy, including
biodiesel, is attracting more interest (Xue et al. 2008;
Ling et al. 2014). Microbially produced oil is a clean
and renewable energy, with short production cycle,
high-lipid content and fatty acid composition similar
to that of common plant oils (Sawangkeaw and
Ngamprasertsith 2013). The restricted development
of microbial oil production in industry is due to the
high costs of raw material. Using cheaper raw material
to produce microbial oil with oleaginous microorgan-
isms is therefore required to solve the problem.
As the industrial wastewater with high COD
(30,000–50,000 mg/L), high ammonium (15,000–
25,000 mg/L) and sulfate (15,000–30,000 mg/L)
concentration and low pH (about 2.0), monosodium
glutamate (MSG) wastewater is one of the most
intractable fermentative wastewaters, and has high
treatment costs when used in conventional activated
sludge processes (Liu et al. 2012). Several studies
have shown that MSG wastewater can be used as a
cheap fermentation broth to produce microbial oil by
fermentation with Rhodotorula glutinis (Xue et al.
2008; Ji et al. 2014). Concentrations of R. glutiniscan reach 9.9 g/L and the lipid content more than
20% after cultivating for 120 h in a basal culture
medium with glucose as the sole carbon source
(Zhang et al. 2014). Research is focused on how to
enhance lipid production. This has shown that high
concentrations of ammonia clearly inhibit cell
growth, but oleaginous microorganisms accumulate
high lipid concentrations under nitrogen-limited
conditions.
The relationship between fatty acid synthesis and
the capacity for lipid accumulation has been studied
(Botham and Ratledge 1979; Wynn and Hamid
1999; Ratledge 2002; Ratledge and Wynn 2002).
Malic enzyme plays a crucial role in lipid accumu-
lation, because NADPH catalytically synthesized by
malic enzyme is the primary reductant for fatty acid
synthesis (Ratledge 2002). The influence of malic
Correspondence: Xu Zhang, National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University of Chemical
Technology, Beijing 100029, China. Tel: +86-10-6445-0593. Fax: +86-10-6441-6428. E-mail: zhangxu@mail.buct.edu.cn
(Received 28 February 2014; revised 4 May 2015; accepted 9 June 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online � 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1201077
enzyme on lipid accumulation in R. glutinis has been
determined, together with the effects of high ammo-
nium sulfate concentrations on lipid accumulation
and malic enzyme activity.
Methods
Strains, culture media, and cultivation conditions
Rhodotorula glutinis (CGMCC No.2258) was sup-
plied by China National Research Institute of Food
and Fermentation Industries. A high lipid yield R.glutinis strain was obtained by ultraviolet mutagen-
esis (Li et al. 2010). After culturing on an agar slant
at 30 �C for three days, the yeast strain was preserved
at 4 �C. The composition of the agar slant was 200 g/
L glucose; 4 g/L yeast extract; 2 g/L urea; 20 g/L agar.
Rhodotorula glutinis was inoculated into the seed
medium (distilled water; 30 g/L glucose; 1.5 g/L
yeast extract; 7 g/L KH2PO4; 0.75 g/L MgSO4;
2 g/L Na2SO4) and grown at 30 �C for 18–24 h.
The wastewater medium was sterilized at 116 �C for
25 min. The composition of the wastewater medium
was similar to the seed medium except that it was
prepared with MSG wastewater and the initial
ammonium sulfate concentrations were 20, 60,
100, 140 g/L. Yeast cultivation was conducted in
250-mL Erlenmeyer flasks at 30 �C on a rotary
shaker (220 rpm) for 96 h.
Measurements of biomass
A 5 mL culture sample was collected every 24 h and
centrifuged at 4800 rpm for 5 min. Afterwards, the
supernatant was removed and the cells were washed
three times with deionized water. Subsequently, the
wet cells were dried to constant weight in a 60 �Coven. The biomass was determined as the dry cell
weight.
Measurements of ammonium sulfate concentration
The concentration of ammonium sulfate was mea-
sured colorimetrically. The principle is as follows:
NH4+ reacts with sodium hypochlorite and then with
phenol in alkaline conditions, catalyzed by sodium
nitroprusside, to produce a blue colored complex
(lmax¼630 nm). The concentration of ammonium
sulfate is proportional to the color of the reaction
solution. A known concentration of 100 mmol/L
ammonium sulfate was prepared as a standard. The
composition of chromogenic reagent I was 10 g/L
phenol; 0.1 g/L sodium nitroprusside. Chromogenic
reagent II contained the solution A (0.4 mol/L
Na2HPO4; 0.2 mol/L NaOH) and B (10% NaClO;
1.8 mol/L NaOH). Dilutions of the standard, 2.5 mL
A solution and 2.5 mL B solution were added into
centrifuge tube in the order stated. After that, the
mixture was kept in a water bath at 37 �C for
45 min and then measured spectrophotometrically
(UV2000, SHIMADZU company, kyoto, Japan).
The linear standard curve provided the relationship
y¼ 10.585x� 0.3482 (R2¼0.9998) (y represents
ammonium sulfate concentration, mmol/L; x repre-
sents OD630).
Lipid extraction and analysis
The total lipid content was measured gravimetrically
every 24 h. Dry cells were ground to a fine powder
and then extracted with a chloroform–methanol
mixture (2:1, v:v) for 3 h (Zhu et al. 2002). The
solvent phase was recovered and the cells were
treated three times in the same way. Finally, the
solvent was evaporated and the total lipid was
obtained. The extracted lipid was treated according
to the methanol esterification procedure, reported by
Guo et a1 (2011).
The lipid fatty acid composition was analyzed by
GC-MS (GCMS-QP2010, SHIMADZU company,
kyoto, Japan) under the following conditions:
Injection volume, 1 mL; Split ratio, 30; Column
oven temperature, 60 �C; Injection temp, 300 �C;
Column DB-5 ms, 30.0 m (length)� 0.25 mm (inner
diameter)� 0.1 mm (thickness); carrier gas, He;
column flow, 1.23 mL/min; temperature program –
initial temperature 60 �C for 1 min increasing at
10 �C/min to 300 �C.
Production of cell extracts
Cells were harvested by centrifugation after 72 h
cultivation, washed three times with distilled water
and suspended in extraction buffer containing
20 mmol/L KCl, 5 mmol/L MnSO4, 2 mmol/L
DTT and 0.1 mmol/L EDTA in 10 mmol/L Tris-
HCl (pH 7.0). After that, the suspension was treated
with a high-pressure homogenizer to obtain the
intracellular cell extracts. Cell debris was removed
by centrifugation at 10,000 rpm for 10 min. The
soluble cell extracts were used to determine the
activity of the malic enzyme. All operations were
carried out on ice (Sukmarinia and Shimizu 2009).
Malic enzyme assays
The activity of malic enzyme was determined using a
double-beam scanning spectrophotometer (UV2000,
SHIMADZU company, kyoto, Japan) equipped with
thermostatic control. The reaction mixture included
Effect of ammonium-N on malic enzyme 19
0.1 mol/L Tris-HCl (pH 7.0), 85 mmol/L MgCl2,
0.6 mmol/L NADP+ and 40 mmol/L malic acid
(Vander et al. 1997). The reaction mixture was put
into a 1-cm light path cuvette and the reaction
initiated by adding the cell extracts to give a final
volume of 500 mL (Peng and Shimizu 2003). Protein
concentrations were determined by the Bradford
method (Bradford 1976).
Results and discussion
Effect of ammonium sulfate concentrations on biomass
The initial ammonium sulfate concentration in the
MSG industrial wastewater was 12.79 g/L. The
concentration of ammonium sulfate in the medium
was adjusted to 20, 60, 100 and 140 g/L by adding
extra ammonium sulfate into the wastewater
medium which was then sterilized and inoculated
with R. glutinis. Figure 1 shows that the ammonium
sulfate concentration had a significant effect on the
R. glutinis biomass produced. In the phase from
0–72 h, the biomass increased continuously with
cultivation time. During the 72–96 h phase, the
biomass still increased at a high rate in the 20 and
60 g/L ammonium sulfate media, but not in the other
two media, in which the biomass had a downward
trend. The maximum biomass concentration at the
end of the fermentation (96h) dropped significantly
from 12 to 6 g/L with an increase in ammonium
sulfate concentration from 20–140 g/L. The results
show that high ammonium sulfate concentrations
inhibits growth but lipid production by R. glutinis is,
nevertheless, an effective approach for the treatment
of MSG industrial wastewater.
Ammonium-N consumption and pH change
The effect of ammonium sulfate concentration on
ammonium-N consumption is shown in Figure 2.
With an increase in initial ammonium concentration,
the uptake of ammonia nitrogen increased and
reached 5.772 g/L at 72 h in flasks containing
140 g/L (Table 1). The pH value of the growth
medium decreased significantly in the first 48 h then
increased slightly in the next 72 h (Figure 3). A
possible reason for the decline in pH value is that
ammonium-N consumption left an increasing
H2SO4 concentration in the fermentation broth or
the metabolism process of R. glutinis produced
extracellular organic acid.
Effects of ammonium sulfate concentration on lipidyield and content
It has been reported that lipid accumulation in
oleaginous microorganisms begins when the nitrogen
is completely used up (Ratledge 2002), however, the
results in this study are quite different. Table.1
shows that although the ammonium sulfate in the
medium was not completely consumed, R. glutiniswas still able to produce lipid. Lipid accumulation
increased slightly with initial ammonium sulfate
concentration over the range from 20 to 140 g/L
and the total lipid yield reached 3.47 g/L when the
initial ammonium sulfate concentration was 60 g/L.
The lipid content was 15.40% at 72 h with initial
ammonium sulfate concentration of 60 g/L and
increased to 21.02% with 100 g/L ammonium sul-
fate. Figure 4 shows the variation of lipid contents
from 0–96 h which shows maximum lipid product-
ivity at 72 h. Lipid accumulation began even when
Figure 2. The residual ammonia nitrogen after cultivation of
R. glutinis in MSG wastewater with various concentrations
of ammonium sulfate at 30 �C. Points are the mean values of
three replicates.
Figure 1. The effect of ammonium sulfate concentration
(20–140 g/L) on growth of R. glutinis in the MSG wastewater.
Points are the mean values of three replicates.
20 G. Gong et al.
high concentrations of ammonium-N were present in
the medium and increased slightly with elevated
amounts of ammonium-N. This phenomenon agrees
with the results of Wu et al. (2010). Total lipid
production (3.47 g/L) and lipid content (15.40%)
were optimum at 60 g/L ammonium sulfate; biomass
productivity was 10.53 g/L (Table 2).
Effect of ammonium sulfate concentration on lipidcomposition
The fatty acid compositions of the lipid from R.glutinis were determined by GC-MS (Table 1).
Lipids from R. glutinis grown on MSG wastewater
contained four main fatty acids, with a composition
similar to that of vegetable oil. The main fatty acids
were C16 and C18; predominantly palmitic acid
(C16:0), linoleic acid (C18:2), oleic acid (C18:1)
and stearic acid (C18:0).
The concentration of ammonium sulfate influ-
enced the fatty acid composition. With increasing
initial ammonium sulfate concentration, the percent-
age of oleic acid increased reaching 76.0% at
140 g/L. Over the range from 20–140 g/L, the
palmitic acid and linoleic acid content decreased
from 21.0–15.4% and from 10.8–4.1%, respectively.
These findings are similar to the previous work (Guo
et al. 2011).
Effect of ammonium sulfate concentration on malicenzyme activity
Malic enzyme plays a vital role in lipid accumulation
by supplying NADPH for fatty acid synthesis
(Ratledge 2002). The total activity of malic enzyme
was measured in extracts from R. glutinis grown with
various levels of ammonium sulfate for 72 h (Table
1). The maximum total activity (18.7 mmol/min) was
obtained at 100 g/L. At this ammonium sulfate
concentration, the maximum lipid content of 21%
was observed (Table.1). Thus, the total activity of
Table 1. The 4(NH4+–N), total lipid yield, lipid contents, composition of lipids, and total malic enzyme activities of R. glutinis cultured in
MSG wastewater with various concentrations of ammonium sulfate for 72 h.
Ammonium sulfate concentration (g/L)
20 60 100 140
4(NH4+–N) (g/L) 0.681�0.10 1.691�0.13 3.197�0.31 5.772�0.45
Total lipid yield (g/L) 3.34�0.08 3.47�0.06 3.00�0.05 2.37�0.09Lipid contents (%) 17.48�0.46 15.40�0.35 21.02�1.46 19.16�0.89Lipid composition (%)Palmitic acid 20.98�0.70 17.84�2.88 17.05�0.83 15.37�0.69Linoleic acid 10.85�1.89 9.77�2.51 6.27�0.71 4.10�0.84Oleic acid 64.24�1.64 67.81�0.02 73.03�1.15 75.97�2.64Stearic acid 3.93�0.76 4.58�0.39 3.65�0.39 4.56�1.11Total activities (mmol/min) 16.929�0.005 16.692�0.001 18.698�0.003 18.162�0.001
All values are the mean of three replicates.
Figure 3. The pH change of R. glutinis cultivated in MSG
wastewater with various concentrations of ammonium sulfate at
30 �C. Values are the mean of three replicates.
Figure 4. The variation in lipid contents of R. glutinis grown in
four different ammonium sulfate concentrations from 0 to 96 h.
Values are the mean of three replicates.
Effect of ammonium-N on malic enzyme 21
malic enzyme correlates with the extent of lipid
accumulation.
Relationship between the lipid content and the malicenzyme activity
Figure 5 shows the relationship between intracellular
lipid content and total malic enzyme activity,
measured after 72 h at various concentrations of
ammonium sulfate. The intracellular lipid content
increased gradually in MSG wastewater cultures with
the increase in malic enzyme activity. The activity of
malic enzyme was high during lipid accumulation
and showed a simple linear correlation (coefficient
0.97) with the intracellular lipid content.
An increase in malic enzyme activity leads to the
accumulation of pyruvic acid within the oleaginous
yeast, which causes an increase in malic dehydro-
genase activity. This converts oxaloacetic acid into
malic acid within the cytoplasm. The consumption
of oxaloacetic acid decreases citric acid production,
causing acetyl-CoA to be diverted to fatty acid
biosynthesis, and derepresses substrate inhibition of
isocitrate lyase. For fatty acid synthesis to occur, it is
not only essential to have a continuous supply of
acetyl-CoA, but also NADPH has to be provided,
primarily via the malic enzyme (Wynn and Hamid
1999; Ratledge 2002). As a result, malic enzyme
remains in a completely active state during the lipid
accumulation phase and promotes the production of
intracellular lipid.
Conclusions
This study has shown that R. glutinis can tolerate
high concentrations of ammonium sulfate. Yeast dry
cell weight could reach 6 g/L after 96 h fermentation
with an ammonium sulfate concentration of 140 g/L.
The highest biomass concentration and total lipid
yield was 10.53 and 3.47 g/L, respectively, with a
lipid content of 15.40% at an ammonium sulfate
concentration of 60 g/L, which would be an optimal
concentration for R. glutinis cultivation in the
wastewater.
We have demonstrated that R. glutinis can accu-
mulate lipids in the presence of high amounts of
ammonium and the concentration of ammonium
influenced the lipid composition. This demonstrates
that it is feasible to produce biodiesel from MSG
wastewater using R. glutinis.
Declaration of interest
The authors report no declarations of interest. The
authors alone are responsible for the content and
writing of the paper. The authors express their
thanks for the support from the National High
Technology Research and Development 863
Program of China (Grant No. 2013AA065804,
2014BAD02B02), the Fundamental Research
Funds for the Central Universities (YS1407).
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Table 2. The effects of ammonium sulfate on growth-related parameters of R. glutinis at 72 h.
(NH4)2SO4 (g/L) X (g/L) L (g/L) Sc (g/L) Y1 (g/g) Y2 (g/g)
20 12.32�0.75 3.34� 0.08 27.60�0.15 0.45�0.04 0.121� 0.0860 10.53�0.51 3.47� 0.06 21.78�0.89 0.48�0.02 0.159� 0.03100 8.12�0.63 3.00� 0.05 19.20�0.12 0.42�0.03 0.156� 0.06140 6.30�0.55 2.37� 0.09 16.74�0.78 0.38�0.01 0.142� 0.02
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Effect of ammonium-N on malic enzyme 23