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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