Journal of Environmental Biotechnology
Vol. 18, No. 1, 69–71, 2018

Technical Note

Dihydroxyacetone Production from Raw Glycerol
by Gluconobacter oxydans NBRC 14819THiroshi Habe1*,†, and Keiji Sakaki1
1

Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST),Tsukuba Central 5–2, 1–1–1 Higashi, Tsukuba, Ibaraki 305–8565, Japan* TEL: +81–29–861–6247 FAX: +81–29–861–8326* E-mail: hiroshi.habe@aist.go.jp†Present address: Environmental Management Research Institute, National Institute of Advanced Industrial Science andTechnology (AIST) (16–1 Onogawa, Tsukuba, Ibaraki 305–8569, Japan)(Received: 18 July, 2017/Accepted: 22 November, 2017)
Key words: biorefinery, raw glycerol, dihydroxyacetone, glyceric acid

Dihydroxyacetone (DHA) is the main ingredient in
self-tanning products 8). DHA is also an important precursor
for chemical synthesis in the pharmaceutical industry 11),and the production of DHA from glycerol using the acetic
acid bacterium Gluconobacter oxydans is cost effective andenvironmentally friendly (Fig. 1). Recent increases in thedemand for DHA have led to an increase in studies focusing
on improved fermentation processes and genetic engineeringof DHA-producing bacteria 7,14). We previously reported thatthe loss of adhA in G. oxydans significantly improved cell
growth, as well as the production of DHA in the presence ofhigh glycerol concentrations 3).
Raw glycerol is a by-product in the biodiesel and oleochemical industries. Approximately 10% of the by-product weightis produced by the transesterification of vegetable oils andanimal fats during biodiesel fuel production 11). Raw glycerol
is purified and used as a feedstock for various industrial
chemicals. However, the purification process is costly andenergy intensive. Therefore, it is necessary to develop methods
for the direct use of raw glycerol, which often contains
methanol and alkali metals. In previous studies, citric acid 9),
mannitol 12), d-arabitol 13), and DHA 2,14) were produced using
raw glycerol; however, raw glycerol (called ‘crude glycerol’in some reports) in those studies contained >80% (w/v)
glycerol and <0.5% (w/v) methanol. Multiple forms ofraw glycerol are produced, depending on the by-production
process 6). Thus, the use of raw glycerol containing more thanFig. 1.

10% (w/v) methanol is important for the development ofrenewable biomass resources.
This study investigated the production of DHA by G.
oxydans (type strain NBRC 14819T). The raw glycerol source(pH 12) was produced via the transesterification of triglycerides (previously provided by Sun Care Fuels Corporation) 1),
and contained 66.4% (w/v) glycerol, 30.9% (w/v) methanol,and 0.54% (w/v) sodium salt (analyzed by our group) 4).
Prior to using the raw glycerol, DHA production by G.
oxydans NBRC 14819T was assessed using pure glycerol ina 1-L jar fermenter (Model MDL; B.E. Marubishi, Tokyo,Japan). For fermentation experiments, five seed cultures intest tubes (5-mL each), which contain both glucose media
and G. oxydans, were aerobically grown for 2 days at 30°C
with agitation at 200 rpm. Seed cultures (total of 25 mL)
were then transferred to a jar fermenter containing 500 mLof glycerol media (pH 6.5), consisting of 100 g/L glycerol,
10 g/L polypeptone, 1 g/L yeast extract, 0.9 g/L KH2PO4,
0.1 g/L K2HPO4, and 1 g/L MgSO4·7H2O, and aerobically
incubated for 3 days. The aeration rate and agitation speed
were set to 1.0 volume of air per volume of medium permin (vvm), and 500 rpm, respectively. The temperature wasmaintained at 30±1°C, and the pH was maintained above 6using 5 M NaOH.
The concentrations of glycerol, DHA, and glyceric acid(GA) in the cultures were quantified using HPLC with an
LC-20AD HPLC pump (flow rate, 1.0 mL/min) and aBioconversion of glycerol to dihydroxyacetone (DHA) and glyceric acid (GA).

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Habe and Sakaki

RID-10A detector (Shimadzu). A Shodex® SC1011 Column(Showa Denko, Tokyo, Japan) was used for glycerol andDHA quantification, while a Shodex® SH1011 Column(Showa Denko) was used for GA quantification. A mobile
phase of pure water was used for the SC1011 column, while
5 mM H2SO4 was used for the SH1011 column. Column
temperatures were maintained at 80°C and 60°C for theSC1011 and SH1011 columns, respectively.
G. oxydans produced 66.2 g/L and 71.8 g/L of DHA
from 100 g/L of pure glycerol following incubations of 2 and3 days, respectively. No glycerol accumulated in the 3-dayculture, while 3.5 g/L of GA did.
Previously, we developed a fed-batch fermentation methodthat used glycerol in an alkaline solution to feed the glycerol and control the pH during GA production 5). Since
raw glycerol contains alkaline metals, including sodium
and potassium, it can be used as an alkaline pH-control
reagent. During DHA production, acids such as GA are
also produced, which reduces the pH of the culture. Thus,
the use of untreated raw glycerol as a pH-control reagent
was investigated during the fed-batch fermentation for DHAproduction.
Since the raw glycerol used in this study could not be usedas the carbon source for Gluconobacter strains without acharcoal pretreatment 4), the effect of the initial pure glycerol
concentration on DHA production was examined using raw
glycerol (pH 12) as the pH-control reagent for maintaining
above pH 6 (Fig. 2). For fermentation experiments, the initial
glycerol concentrations in the glycerol media were 5, 10, or
20 g/L. For media containing 20 g/L initial glycerol, 82.9 g/Lof DHA and 4.3 g/L of GA were produced during the3-day incubation (Fig. 2A). Glycerol contents were increased
to 83.6 g/L because the feeding rate of the raw glycerol
exceeded the consumption rate during growth and DHA
production. Incubations up to 5 days did not increase DHA
concentrations in the cultures. Following 3-day incubations
with starting glycerol concentrations of 10 g/L and 5 g/L,
74.0 and 75.2 g/L of DHA and 6.7 and 5.0 g/L of GA wereproduced, respectively (Figs. 2B and 2C). A gradual accumulation of glycerol was also observed in the culture medium forboth experiments. Decreasing the initial glycerol concentration to <5 g/L resulted in poor growth (data not shown).
The culture conditions for DHA production by G. oxydans
were optimized for an initial pure glycerol concentration of
5 g/L. Figure 3A shows the time course of DHA production
using untreated raw glycerol with an aeration rate of 2 vvm(pH 6), and 86.9 g/L of DHA was produced over a 3-dayincubation. When the agitation speed was increased from500 to 700 rpm, DHA production increased to 105.1 g/Lover the 3-day incubation (Fig. 3B). Thus, DHA production
with concentrations of >100 g/L can be achieved using raw
glycerol feedstock. Notably, GA production ranged from 3.4
to 6.6 g/L in all experiments.
We previously showed that methanol decreased GA production by G. oxydans because it inhibited membrane-boundalcohol dehydrogenase activity, which catalyze the oxidationof glycerol to glyceraldehyde 10). In this study, low productionof GA was also observed probably because of the presenceof methanol in raw glycerol. By contrast, methanol did notinhibit glycerol dehydrogenase, which converts glycerol toFig. 2. Time course of DHA production by Gluconobacter oxydans NBRC14819T with initial glycerol concentrations of (A) 20 g/L, (B)
10 g/L, and (C) 5 g/L. Experiments were performed using a 1-L jar fermenter. Cultures were maintained at a pH of 6 using untreated
raw glycerol (pH 12). An aeration rate of 1.0 vvm and an agitation speed of 500 rpm were used. Symbols: black circles, glycerol concentration; white squares, DHA concentration; white circles, GA concentration; black squares, dry cell weight.

Dihydroxyacetone Production from Raw Glycerol by Gluconobacter oxydans NBRC 14819TFig. 3. DHA production by Gluconobacter oxydans NBRC14819T
during 3-day incubations with initial glycerol concentrations of
5 g/L. Experiments were performed using a 1-L jar fermenterwith (A) a 2.0-vvm aeration rate and 500-rpm agitation speed,or (B) a 2.0-vvm aeration rate and 700-rpm agitation speed.
The pH was maintained at 6 using untreated raw glycerol(pH 12). Symbols: black circles, glycerol concentration; whitesquares, DHA concentration; white circles, GA concentration;
black squares, dry cell weight.

DHA. Therefore, DHA can be produced from untreated raw
glycerol containing methanol. However, further improvements should be made to the yield and purification of DHA.
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