Journal of Bioscience and Bioengineering

VOL. 111 No. 6, 658 – 664, 2011

www.elsevier.com/locate/jbiosc

Production of biomass and extracellular 5-aminolevulinic acid by Rhodopseudomonas

palustris KG31 under light and dark conditions using volatile fatty acid

Wanna Choorit,1,⁎ Angkana Saikeur,2 Pichit Chodok,1 Poonsuk Prasertsan,3 and Duangporn Kantachote4

School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80160, Thailand, 1 Faculty of Science and Technology,
Rajamangala University of Technology, Nakhon Si Thammarat 80240, Thailand, 2 Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112, Thailand, 3
and Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand 4

Received 24 August 2010; accepted 26 January 2011

Available online 24 February 2011

Kinetic parameters for growth and extracellular 5-aminolevulinic acid (ALA) production of Rhodopseudomonas palustris
KG31 under light and dark conditions in a medium containing volatile fatty acids (VFA) as the carbon sources were estimated
using a Gompertz model. The lag phase for growth and the maximum specific growth rate under microaerobic-light
cultivations were 7.29–12.49 h and 0.038–0.094 h− 1, respectively, whereas under aerobic-dark cultivations, they were 2.03–
14.25 h and 0.016–0.022 h− 1, respectively. The lag phase for extracellular ALA production and the maximum specific
extracellular ALA production rate under microaerobic-light cultivations (15.72–24.74 h and 0.222–0.299 h− 1, respectively)
were better than those obtained under aerobic-dark cultivations (24.57–44.84 h and 0.103–0.215 h− 1, respectively). The
biomass and the extracellular ALA yields of 39.66–56.25 gDCW/l/molC, and 148.47–245.75 μM/molC, respectively, under
microaerobic-light cultivations were higher than of those obtained under aerobic-dark conditions. An enhancement of
extracellular ALA production under aerobic-dark conditions revealed that the ALA yield was markedly increased 8-fold
(48.36 μM) by the addition of 10 mM succinate, 4.5 mM glycine, and 15 mM levulinic acid (LA). By controlling dissolved oxygen
(DO) and pH values, a maximum extracellular ALA yield of 66.38 μM was found. The degradation rate of ALA in the culture
broth was closely related to the pH value.
© 2011, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Aerobic-dark; Gompertz model; Microaerobic-light; Photosynthetic bacteria; Volatile fatty acids]

5-Aminolevulinic acid (ALA) is an intermediate metabolite
generated and utilized for the biosynthesis of tetrapyrroles such as
heme, chlorophyll, and vitamin B12 in animals, plants, algae, and
bacteria. In photosynthetic bacteria, it is synthesized via the Shemin
pathway, involving the two precursors; succinyl-CoA and glycine
which are catalyzed by aminolevulinic synthase (ALAS). The step after
ALA formation is a porphobilinogen (PBG) synthesis via the action of
aminolevulinic dehydratase (ALAD) (1–3).
ALA is an effective and non-toxic biodegradable herbicide and
insecticide. It can also be used as a plant growth promoter under
normal and stress conditions (4–11). As reported by Nishihara et al.
(6) plants treated with 0.60 and 1.80 mmol/l ALA showed significant
increases in the photosynthetic rate at 50 and 100 mmol/l NaCl. It also
improved salt tolerance growth of date palm seedlings by increasing
photosynthetic assimilation (9). Apart from ALA formation, the
photosynthetic bacterial cells are also able to fix atmospheric nitrogen
as their cells are rich in pigments (carotenoids). Therefore, application
⁎ Corresponding author. Tel.: +66 75 672355; fax: +66 75 672302.
E-mail address: cwanna35@gmail.com (W. Choorit).
of ALA and the biomass of photosynthetic bacteria in the agricultural
field are potentially very useful.
Photosynthetic bacteria grow and produce ALA under microaerobic-
light and aerobic-dark conditions. Tanaka et al. (12) reported that
Rhodobacter sphaeroides CR-17 produced ALA under microaerobic-dark
conditions, 64.5 μM – 2-fold higher than obtained under aerobic-dark
conditions. An expression of the hemA encoded for ALAS in Rhodobacter
capsulatus grown under semiaerobic conditions was about 40% of that
under photosynthetic conditions while expression of the hemB encoded
for ALAD under semiaerobic was about 80% of that under photosynthetic
conditions (13). Meanwhile, photosynthetic growth of Rhodobacter
capsulatus resulted in higher ALA production than that of semiaerobic
growth.
We have found that Rhodopseudomonas palustris KG31 posses some
interesting properties such as an acid tolerance and utilizes volatile fatty
acids (VFA) as a source of carbon. These properties are very useful for
enhancing ALA yield under heterotrophic growth (2,14). For example at
low pH, levulinic acid (LA) in undissociated form was effectively utilized
by cells (14). This also implied that a high amount of ALA remained in
the culture medium due to ALAD activity being drastically inhibited.
In this work the Gompertz model was used to better understand
kinetic parameters for growth and ALA production by R. palustris

1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2011.01.014
VOL. 111, 2011 BIOMASS AND ALA PRODUCTION UNDER LIGHT AND DARK CONDITIONS
KG31 grown in a medium containing different types of VFA (acetic
acid, propionic acid and butyric acid) under light and dark conditions.
This model was first described by Zwietering et al. (15). The criterion
for application of the model is a sigmoidal curve of bacteria, beginning
with a lag phase and then growth is accelerated to a maximal value in
a certain period of time. After that growth reaches a final phase, called
an asymptote. The model is generally accepted to be applicable for
calculation of kinetic parameters for growth and the growth
associated product (16–18). The process for using this model
comprises two steps: (i) gathering growth and growth associated
product data from the beginning of the experiments over a range of
interest and (ii) testing the data with the model.
A Box–Behnken design and a response surface method (RSM)
were used to determine the concentrations of succinate, glycine, and
LA added to the VFA medium which optimized the ALA yield under
aerobic-dark conditions. The Box–Behnken design is an effective
screening design. The RSM is a statistical method that uses
quantitative data from appropriate screening design (i.e., Box–
Behnken design) to determine (i) the factor levels that will
simultaneously satisfy a set of desired specifications, (ii) the optimum
combination of factors that yield a desired response and describes the
response near the optimum, and (iii) how a specific response is
affected by changes in the level of the factors over the specified levels
of interest (19). Based on the results of the RSM, experiments in a
reactor were carried out to investigate the effect of dissolved oxygen
(DO) and pH on ALA production. Therefore, this paper aimed to
illustrate kinetic parameters for growth and ALA production by R.
palustris KG31 under light and dark cultivations using VFA as a carbon
source and determine the factors (succinate, glycine and LA) that
influence extracellular ALA production, and evaluate ALA stability
under various pH values.
MATERIALS AND METHODS
The bacterium The photosynthetic bacterium, R. palustris KG31 was isolated
from soil collected from a paddy field in Nakhon Si Thammarat province, Thailand, and
identified using morphological, biochemical, and physiological properties and 16 s
rDNA sequencing by Pichit Chodok, a master student of the Biotechnology Program,
Walailak University, Thailand.
Chemical and media A GM medium was used to maintain the bacterial cells and
to prepare a starter culture. The medium contained 0.5 g/l of yeast extract, 2.7 g/l of
malate, 3.7 g/l of monosodium glutamate, 0.8 g/l of (NH4)2SO4·7H2O, 0.5 g/l of KH2PO4,
0.5 g/l of K2HPO4, 0.2 g/l of MgSO4·7H2O, 1.0 × 10− 3 g/l of thiamine–HCl, 1.0 × 10− 3 g/l of
nicotinic acid and 1.0 × 10− 5 g/l of biotin.
Since in an anaerobic treatment, a mixed volatile fatty acid that is acetic acid,
propionic acid and butyric acid were found in a concentration of 1.80 g/l, 0.2 g/l, and
1.0 g/l (20). In order to investigate volatile fatty acid utilization by the bacterium, acetic
acid, propionic acid or butyric acid is added to GA, GP and GB media, respectively. GA,
GP, GB and VFA media, used as test media, had a composition similar to the GM
medium, except that the yeast extract was 0.2 g/l, DL-malic acid was not added, but
2.42 g/l of acetic acid, 1.99 g/l of propionic acid and 1.77 g/l of butyric acid (equal to
0.08 mol carbon) were added to GA, GP and GB media, respectively. In the VFA medium,
1.80 g/l acetic acid, 0.2 g/l propionic acid and 1.0 g/l butyric acid were added instead of
DL-malic acid. The initial pH of the media was adjusted to 6.5 prior to heating in an
autoclave.
Starter culture preparation The bacterium was cultivated in 375-ml flat vessels
containing 350-ml the GM medium, and incubated at 35 ± 1°C under microaerobic-light
conditions (static-light). This was achieved by illumination of the surface of the vessel
(14.64 W/m2; using the factor of 1 W/m2 = 683 lux) (21) with a tungsten bulb, and
incubation of the vessel under static conditions with gently mixing to produce a
homogenous mixture during cultivation by stirring the medium at 70 rev/min with a
magnetic bar. After growth reached an early stationary phase, the bacterium was
harvested by centrifugation at 5590×g for 15 min. The cell pellet was washed with 0.9%
NaCl to remove nutrients and resuspended in a sterilized culture medium as desired (will
be provided later). The cell concentration was adjusted to an optical density (OD660 nm) of
1.0 by using a spectrophotometer (UV-1600 UV–VIS Spectrophotometer, Shimadzu,
Japan). The starter culture of 10% was inoculated into the medium.
Biomass and extracellular ALA production under light and dark cultivations
Profiles of growth and extracellular ALA production were performed by cultivating
the bacterium in GA, GP, GB and VFA media under microaerobic-light and aerobic-dark
conditions, at 35 ± 1°C. The microaerobic-light conditions were achieved by placing
1.5-l of the medium into a 1.6-l Roux bottle, and cultivated under the conditions as
659
described above. The aerobic-dark condition was performed in a 1-l Erlenmeyer flask
containing 0.45-l of the medium, covered with black vinyl and shaken on a rotary
shaker (Gallenkamp, Sanyo, Japan) at 150 rev/min. Bacterial growth and extracellular
ALA were measured every 4 h until a stationary phase was observed.
Enhancement of extracellular ALA production The experiment was carried
out by inoculating 20% of the starter culture into a 1-l Erlenmeyer flask containing 0.45-
l VFA medium plus succinate, glycine and LA; they were added to the VFA medium at
three levels (low, medium, and high) and coded (− 1, 0 and + 1) (Table 3), according to
the Box–Behnken design (22). Succinate and glycine were added at time 0 h; then LA
was added after 24 h cultivation. The design consisted of triplicates at the center point
and points as shown in Table 3. The experiment plan consisting of 15 runs were carried
out in duplicate. In order to answer the most suitable conditions and effect of each
independent variable toward ALA production, response surface method was employed.
The three independent variables (succinate, glycine and LA) selected for the statistical
analysis were designated X1, X2 and X3 respectively. The predicted response, Y stands
for ALA yield. First, the data obtained from the Box–Behnken experiments were entered
into the Design-Expert Software Trial Version (Stat-Ease, Minneapolis, MN, USA). This
software contains sequential techniques that were used for evaluation of the effect of
the variables on the response. Secondly, selecting an appropriate type of model for the
response was done by testing the sequential F-tests, starting with a linear model and
adding terms (quadratic, and higher if appropriate). Finally, the F-statistic was
calculated for each model, and the highest order model with significant terms was
chosen. Tests for significant sequential models, model equation, and model terms were
performed by employing analysis of variance (ANOVA). The quality of fit of the model
equation was expressed by the coefficient of determination (R2) and adjusted R2. Using the
Design-Expert Software, the optimal values of the ALA of the experimental conditions were
obtained by analyzing the response surface contour plots. Then, the response surface at
optimal points was selected. As for predicting the optimal point of the experiment, a
quadratic second order polynomial model was fitted to correlate the relationship between
independent variables and the response. The quadratic second order polynomial equation
was as follows:
Y = β0 + β1 X1 + β2 X2 + β3 X3 + β12 X1 X2 + β13 X1 X3 + β23 X2 X3 + β11 X12
+ β22 X22 + β33 X32 : ð1Þ
The coefficient, β0 is a model constant called the intercept. The terms X1, X2, and X3
are independent variables; β1, β2, and β3 are linear coefficients; β12, β13, and β23 are
cross-product coefficients, and β11, β22, and β33 are the quadratic coefficients.
Bioreactors experiment Bioreactor (Bioflo 110, New Brunswick Scientific, NJ,
USA) experiments were performed by inoculation of 20% starter cultures with an
optical density of 1 to 3-l containing 2.1-l of VFA medium with the most suitable
concentrations of succinate, glycine, and LA from the previous experiments. Four runs,
A–D, were performed. Run A, DO and pH were throughout controlled at 5% and 6.5–7.0,
respectively. Run B, DO and pH were throughout controlled at 1% and 6.5–7.0,
respectively. Run C, DO was controlled at 5% from 0 to 16 h of operation times, and was
adjusted to 1% onwards whereas pH was throughout kept at 6.5–7.0. Run D, DO and pH
were controlled at 5% and 6.5–7.0 from 0 to 16 h of operation times, and adjusted to 1%
and 6.0–6.5 onwards, respectively. Oxygen and nitrogen gases were automatically fed
to the culture broth to obtain the desirable DO levels. DO concentrations were
controlled by automatic oxygen control devices. The pH ranges of the cultures were
controlled by pH-stat and automatically adjusted with 10% NaOH and 20% H3PO4. In run
D, the pH was adjusted to 6.0–6.5 after LA was added. Temperature and agitation speed
were set at 35°C and 150 rev/min, respectively.
Stability of ALA This experiment was performed by taking 10-ml of the culture
broth containing ALA at 12.04 μM and the bacterial cells at a density of 8.7 × 108 c.f.u./ml
(run no. 8) and placed in test tubes. Then, the pH of each test tube was adjusted using
1 N HCl and 1 N NaOH solutions as desired, and stored in the dark at 4°C (refrigerator)
and 30°C (incubator room). The initial and final concentrations of ALA and the bacterial
cells at 0 and 30 days were determined.
Analytical methods Biomass was measured as turbidity using a spectropho-
tometer (UV-1601, Shimadzu, Kyoto, Japan) at a wavelength of 660 nm and correlated
with the dry cell weight (DCW) measured by drying the cells at 105°C for 12 h.
Extracellular ALA was determined by a colorimetric method (23). The residual VFA, LA
and succinate were determined with HPLC (2690, Waters, MA, USA) with a reflective
index detector (2414, Waters, MA, USA). The HPLC conditions were as follows: column:
metaCarb H plus column (Varian, CA, USA); temperature: 68°C; mobile phase: 8.5 mM
H2SO4; and flow rate 0.4 ml/min. The concentrations of residual VFA, LA and succinate
were calculated from the peak areas. The pH value was measured using a pH meter
(CyberScan pH 510, Ayer Rajah Crescent, Singapore).
All curves were fitted by a sigmoidal model (Gompertz equation). The Gompertz
equation is written as:
Y ¼ a · eð−exp½−cðt−mÞÞ
For each curve Y is the amount of biomass or extracellular ALA, at time t (h); a is the
asymptotic amount of products that occurs as time increases indefinitely. The first order
derivate represents the production rate of biomass, and extracellular ALA; c is the relative
production rate at m, where m is the time at which the production rate is maximized. A
maximum specific growth rate, a maximum specific extracellular ALA production rate, a
660 CHOORIT ET AL. J. BIOSCI. BIOENG.,

lag phase for growth and a lag phase for extracellular ALA production were calculated using
the following equations (15).
μ m = ac = e
λ = m−ð1 = cÞ
Viable counts of R. palustris KG31 cells were obtained on plates containing the G5
medium (24). The plates were inoculated with the appropriate diluted R. palustris
KG31, incubated under aerobic and dark conditions, at 37°C for 36 h. Degradation rate
of ALA (%DR) was calculated using the following equation:
%DR = ½ðALAt = 0 –ALAt = 30 Þ × 100 = ALAt = 0
where ALAt = 0 and ALAt = 30 are ALA concentrations at day 0 and at day 30, respectively.
RESULTS AND DISCUSSION
Biomass and extracellular ALA production under light and
dark cultivations The model gave good fits with the experimental
data of the bacterial growth and the extracellular ALA production with
R2 of 0.94–0.99. Different kinetic parameters of growth and extracel-
lular ALA production of the bacterium are indicated in Table 1.
A lag phase for growth and a maximum specific growth rate are the
most studied in the fermentation process. A lag phase for growth is
determined by the time needed by the bacteria to adapt their
metabolism routes to the surrounding media to start the exponential
growth until the maximum population is reached in the environment.
Comparisons of the lag phases for growth revealed the bacterium
seems to be adjusted its metabolism to acetic acid, propionic acid and
butyric acid under aerobic-dark conditions better than under
microaerobic-light conditions. Under aerobic-dark conditions in
propionic acid, the lag phase for growth of 7.97 h was observed
whereas in acetic acid and butyric acid, the lag phases for growth were
2.03 h and 3.66 h, respectively. While the lag phase for growth under
microaerobic-light conditions in acetic acid, propionic acid and
butyric acid was 7.29, 12.49 and 7.67 h, respectively. The time taken
to alter metabolic pathways were longer but the maximum specific
growth rate of the bacterium in microaerobic-light cultivations
(0.038–0.072 h− 1) was faster than under aerobic-dark conditions
(0.016–0.022 h− 1).
At the end of the cultivation time, the maximum biomass under
microaerobic-light conditions was higher than under aerobic-dark
conditions. Under microaerobic-light condition the biomass in acetic
acid was 3.0 gDCW/l, 3-fold higher in comparison with under aerobic-
dark (0.9 gDCW/l) conditions (Table 1). In numerous studies, the
availability of light and DO tension have been regarded as the major
factors affecting the growth and viability of anoxygenic photosyn-
thetic bacteria (25). The results above agree with those obtained from
Rhodovulum sulfidophilum (cultivated in non-sterilized sardine pro-
cessing wastewater) (26), Rhodobacter sphaeroides S and Rhodovulum
sp. PS88 in GMS medium (27), Rhodomicrobium vannielii (28) as well
as Rhodobium sp. NW16 and Rhodobacter sp. KMS24 (29).
Table 1 shows that the maximum specific extracellular ALA
production rate under microaerobic-light cultivation was about 2-
ð2Þ fold faster than of those cultivated in aerobic-dark conditions. Under
aerobic-dark conditions, the lag phase for extracellular ALA produc-
tion was in the range of 24.57–44.84 h whereas it took a shorter time
ð3Þ
under microaerobic-light conditions. Under aerobic-dark conditions
in acetic acid, the maximum specific extracellular ALA production rate
was 0.215 h− 1 with a lag phase of 26.21 h. These values were similar
to those obtained under microaerobic-light condition in propionic
acid. The maximum extracellular ALA production obtained under
microaerobic-light conditions (11.70–13.06 μM) was about two to
ð4Þ
three times higher that under aerobic-dark conditions (4.76–
7.99 μM).
Comparison of the biomass yield, the extracellular ALA yield and
the carbon utilization of the bacterium grown under microaerobic-
light and aerobic-dark conditions are shown in Table 2. The highest
biomass yield (56.25 gDCW/l/molC) and extracellular ALA yield
(245.75 μM/molC) were achieved under microaerobic-light condi-
tions in butyric acid as the carbon source. The lowest yields of biomass
(16.98 gDCW/l/molC) and the extracellular ALA (89.81 μM/molC)
were found under aerobic-dark condition when acetic acid was the
carbon source. The biomass yield and extracellular ALA yield obtained
under microaerobic-light conditions were significantly different from
those found in aerobic-dark conditions. Thus it appears that R. palustris
KG31 prefers to grow and produce ALA under light conditions as
compared with dark conditions.
Acetic acid utilization by the bacterium under microaerobic-light
conditions was a little bit higher than under aerobic-dark conditions.
Propionic acid and butyric acid were completely exhausted under
aerobic-dark cultivations (Table 2). During cultivation, we found
acetic acid in the media when propionic acid and butyric acid were
added as the carbon sources. Wang et al. (30) reported that
degradation of propionic acid produced acetate, according to the
following equation:
CH3 CH2 COOH + 3H2 O → CH3 COOH + HCO− þ
3 + H + 2H2
Fang et al. (31) reported that the degradation of butyrate produced
acetate, as illustrated in the following equation:
CH3 ðCH2 Þ2 COOH + 2H2 O → 2CH3 COOH + 2H2
Therefore the bacterium secreted an extracellular enzyme(s) to
hydrolyze propionic acid and butyric acid to acetic acid. This led to an
extension of a lag phase for growth in propionic acid and butyric acid
under both conditions (Table 1). Thus, the C2-unit enters the central
carbon metabolism on the level of acetyl-CoA which is assimilated via
glyoxylate cycle (32,33). Unlike R. sphaeroides which assimilates
propionate by the methylmalonyl-CoA pathway (34).
The results of biomass yield obtained under microaerobic-light
conditions are fairly high. This can explained due to the presence of
acetic acid under photoheterotrophic conditions of R. palustris, the
Calvin cycle functions to effectively balance the redox potential of
the cell by allowing CO2 to be utilized as an electron acceptor for the

TABLE 1. Comparisons of lag phase (λ) and maximum specific (μm) rate for growth and extracellular ALA production under light and dark conditions by R. palustris KG31.
Conditions Carbon Growth Extracellular ALA
sources
Biomass (g DCW/l) Max biomass (h) λ (h) μm (h− 1) R2 ALA (μM) Max ALA (h) λ (h) μm (h− 1) R2

Light Acetic acid 3.0 92 7.29 0.072 0.99 12.45 80 16.91 0.299 0.99
Propionic acid 2.3 88 12.49 0.038 0.99 13.06 80 24.74 0.222 0.96
Butyric acid 2.7 84 7.67 0.071 0.99 11.70 76 16.17 0.252 0.98
Mixed VFA 3.6 92 10.47 0.094 0.99 12.62 76 15.72 0.297 0.99
Dark Acetic acid 0.9 68 2.03 0.019 0.96 4.76 60 26.21 0.215 0.95
Propionic acid 1.8 80 7.97 0.016 0.97 6.87 84 24.57 0.103 0.97
Butyric acid 1.8 84 3.66 0.022 0.99 7.99 84 37.59 0.107 0.94
Mixed VFA 1.7 92 14.25 0.021 0.99 6.56 88 44.84 0.126 0.95
VOL. 111, 2011 BIOMASS AND ALA PRODUCTION UNDER LIGHT AND DARK CONDITIONS 661

TABLE 2. Comparison of biomass yield, extracellular ALA yield and carbon utilization by TABLE 4. Analysis of variance (ANOVA) for the fitted quadratic model for optimization
R. palustris KG31 grown under light and aerobic-dark conditions. of ALA production.
Conditions Carbon sources Biomass yield Ext. ALA yield Carbon Source df SS MS P
(g DCW/l/molC*) (μM/molC*) utilization (%)
Model 9 1622.23 180.25 0.0304
Light Acetic acid 46.88 194.53 79.01 Residual 5 147.91 29.58
Propionic acid 39.66 225.17 71.60 Lack of fit 3 139.81 46.60 0.0810
Butyric acid 56.25 245.75 60.00 Pure error 2 8.10 4.05
Mixed VFA 42.35 148.47 73.28 Corrected total 14 1770.14
Dark Acetic acid 16.98 89.81 65.43
R2 = 0.9164, adjusted R2 = 0.7660.
Propionic acid 22.22 84.81 100
Butyric acid 22.22 99.88 100
Mixed VFA 16.35 63.08 89.66

*Based on carbon sources (acetic acid, propionic acid and butyric acid) to be added in
the medium; Each mole of acetic acid, propionic acid and butyric acid contains 3, 4 and succinate, 10 mM glycine and 10 mM LA were added, 9.08 μM ALA
5 mol carbons, respectively. was produced (run no. 15). However, when the precursors (succinate
and glycine) and the inhibitor (LA) were not added, the bacterium
produced 6.56 μM of ALA. Addition of the agents in the medium
removal of excess reducing equivalents which are generated from the
therefore has a significant effect on extracellular ALA production.
oxidation of organic electron donor/carbon source supplied to the cell.
The results of the 15 runs were investigated to find the best model
The activity of ribulose 1,5-bisphosphate (RuBP) carboxylase, the
to represent the maximum concentration of extracellular ALA by
primary catalyst of CO2 fixation in photosynthesis, present under
entering the results of the experimental data obtained from the Box–
photoautotrophic conditions, was repressed by chemoheterotrophic
Behnken design to the Design-Expert Software. An ANOVA table
growth but was not decreased by the presence of organic substrates
showed that the quadratic model is the highest order model with
during photoheterotrophic growth (32). During the aerobic-dark
significant terms (P = 0.0304) (Table 4). The regression equation for
conditions, the main part of the carbon source is completely oxidized
extracellular ALA production by the bacterium was therefore given as
in the TCA cycle, and only a minor part is assimilated as intracellular
follows:
material (35).
The mixed VFA of these concentrations was used to evaluate Y ðμMÞ = 37:58 + ð5:24X1 Þ−ð6:67X2 Þ + ð3:92X3 Þ + ð3:42X1 X2 Þ
growth, extracellular ALA production by the bacterium. Table 1 shows




the lag phase for growth was 10.47 h and 14.25 h in microaerobic- + ð10:26X1 X3 Þ + ð1:10X2 X3 Þ− 5:75X12 − 9:66X22 − 3:00X32
light and aerobic-dark cultivations, respectively. The maximum ð5Þ
specific growth rates 0.094 h− 1 and 0.021 h− 1 were observed in
microaerobic-light and aerobic-dark cultivations, respectively. The lag where the variables take their coded values, and represent extracellular
phase for extracellular ALA was 44.84 h for the aerobic-dark culture. ALA yield (Y) as a function of succinate (X1), glycine (X2), and LA (X3).
Biomass yield and extracellular ALA yield of the bacterium grown The maximum extracellular ALA yield obtained from the predic-
under microaerobic-light conditions were 42.35 gDCW/l/molC and tion was 48.23 μM (run no.12) (Table 3). Eq. 5 indicated that in order
148.47 μM/molC, respectively, whereas they were 16.35 gDCW/l/ to enhance extracellular ALA production, succinate, and LA should be
molC and 63.08 μM/molC, respectively. Carbon utilization under supplied in the medium. The soundness of the model can be checked
microaerobic-light condition (73.28%) was a little bit lower than by the determination coefficient, R2 and the adjusted R2. The value of
under aerobic-dark conditions (89.66%) (Table 2). adjusted R2 suggested that the total variation of 91.64% for the
Enhancement of extracellular ALA production under dark extracellular ALA was to be attributed to the independent variables
conditions The data in Table 3 shows the variation of ALA yields and about 8.46% of the total variation could not be explained by the
(9.08-49.29 μM) obtained in the 15 runs. This variation implied the model. The value of lack-of-fit for the regression equation indicated
influence of succinate, glycine, and LA on extracellular ALA production that the model was adequate for predicting the yield of extracellular
by R. palustris KG31 under aerobic-dark conditions. The addition of ALA under any combination of values of the variables (Table 4). The
10 mM succinate, 5 mM glycine, and 15 mM LA to the VFA medium coefficient estimates of Eq. 5 and the corresponding P-values are
(run no. 12) increased the amount of ALA markedly. When 0 mM shown in Table 5. P-values of variables β2 and β13 were 0.0178
and 0.0130, respectively. Eq. 5 and the P-value of variable β2 clearly
indicate a significant negative effect of glycine while Eq. 5 and the P-
TABLE 3. Box-Behnken design with the actual and predicted values of ALA production
value of variable β13 clearly show a significant positive effect of
by R. palustris KG31. succinate and LA on extracellular ALA production.
Run Design ALA at 48 h (μM) A contour curve was drawn by fixing the glycine concentration at
5 mM, so that the interaction between succinate and LA on the
X1 X2 X3 Actual Predicted
extracellular ALA yield during cultivation of the bacterium for 48 h
1 5 0 5 25.41 28.77
2 5 10 15 26.63 23.30
3 10 10 10 19.74 24.21
4 0 5 15 11.63 17.25 TABLE 5. Coefficient of the response function to predicted ALA formation.
5 5 5 10 39.90 37.59
Variable Coefficient Standard error F P
6 10 5 5 25.51 19.92
7 5 5 10 36.31 37.59 β0 37.58 3.14 6.09 0.0304
8 5 10 5 12.04 13.29 β1 5.24 1.92 7.43 0.0415
9 10 0 10 28.42 30.64 β2 − 6.67 1.92 12.04 0.0178
10 5 5 10 36.53 37.59 β3 3.92 1.92 4.15 0.0974
11 5 0 15 35.61 34.38 β12 3.42 2.72 1.58 0.2644
12 10 5 15 49.29 48.23 β13 10.26 2.72 14.23 0.0130
13 0 0 10 31.43 27.01 β23 1.10 2.72 0.16 0.7032
14 0 5 5 28.88 29.94 β11 − 5.75 2.83 4.13 0.0978
15 0 10 10 9.08 6.88 β22 − 9.66 2.83 11.64 0.0190
β33 − 3.00 2.83 1.12 0.3379
The data in the table under “Actual” represent means of duplicate experiments.
662 CHOORIT ET AL.
FIG. 1. Contour plot shows the effect of succinate and LA toward ALA yields.
was obtained (Fig. 1). Addition of succinate and LA resulted in an
increase in ALA. From Eq. 5, and the contour curve addition of 10 mM
succinate, 4.5 mM glycine and 15 mM LA gave 48.36 μM ALA.
Note that, the experiments for enhancement of ALA production by
the bacterium revealed that succinate was a limiting factor under
FIG. 2. ALA yields (filled symbols) and biomass yields (open symbols) of R. palustris KG31 in the VFA medium supplementation with 10 mM succinate, 4.5 mM glycine, and 15 mM LA.
The arrow indicates the time-point at which the LA was added. The dotted line indicates the time-point at which the dissolved oxygen and the pH values were changed. The reactor
operated under the conditions as were described in Materials and Methods.
J. BIOSCI. BIOENG.,
aerobic-dark conditions while glycine was a limiting factor under
microaerobic-light conditions (23). This can be explained because
under aerobic-dark conditions, succinate is rapidly used in the TCA
cycle while glycine may be used as an electron donor or a precursor
for producing macromolecules such as purine under microaerobic-
light conditions. LA gave positive results on extracellular ALA
production under both conditions. However, optimal concentrations
of succinate, glycine, and LA added to culture media of the both
conditions differed. The maximum ALA yield was 134.47 μM obtained
by growing the bacterium under microaerobic-light conditions in the
VFA medium supplemented with 5 mM glycine and 10 mM LA (23).
However, under aerobic-dark conditions the bacterium required
10 mM succinate, 5 mM glycine and 15 mM LA to produce 49.29 μM
extracellular ALA. Taking these results, the precursors and the
inhibitor did drastically affect ALA production under microaerobic-
light conditions more than under aerobic-dark conditions. Corre-
spondingly, ALA production under aerobic-dark condition was about
37% of that found for microaerobic-light conditions. As reported by
Wang et al. (36) ALA production by Escherichia coli harbored hemA of
R. sphaeroides was increased after adding glycine. Rhodobacter
sphaeroides CR-606 accumulated 11.2 mM ALA in the presence of
50 mM glucose, 60 mM glycine, 5 mM LA, and yeast extract under
conditions of agitation in the absence of light (37). Recombinant E. coli
harboring a fusion gene hemA:egfp required 30 mM glucose, 15 mM
glycine, 30 mM succinic acid, and 30 mM LA to produce 1000 mg/l ALA
(38). These results indicated that bacterial strain, medium composition
and cultivation condition affected metabolites for ALA production.
Bioreactors experiment Concerning the above results and
those information that the maximal activity of ALAS occurred at pH
VOL. 111, 2011
6.5 (39), the effect of high DO significantly decreased the expression
of hemA which encoded ALAS activity (13) Thus, investigations on the
effect of DO concentrations and pH on biomass and extracellular
ALA production were carried out. The VFA medium was supplemented
with 10 mM succinate, 4.5 mM glycine and 15 mM LA. In all
experiments, the DO probe was set at desirable values. However,
the DO recorder showed about ± 0.5 deviation.
Fig. 2 shows extracellular ALA contents and cell growth. Growth rate
of the bacterium in run A was faster than that in run B. At 20 h, the
biomass of runs A and B were 2.8 and 1.2 gDCW/l, respectively. In run A,
a lag phase was observed after the addition of LA. This indicated that the
bacterial cells adjusted their metabolic pathways for the incoming
substance when the amount of DO was enough for growing cells. At
40 h, the biomass of runs A and B were 4.2 gDCW/l (Figs. 2A and 2B).
When operating the bioreactor under the conditions identical to A and B
but without LA the maximum biomass was 2.5 gDCW/l (data not
shown). Thus, LA serves not only as a competitive inhibitor of ALA, but
also as the carbon source for cell growth. The effectiveness of LA as the
competitive inhibitor of ALA was observed in run B where the DO was
1%. At 32 h, the amount of extracellular ALA was 61.38 μM (Fig. 2B).
Adjusting the pH to 6.0–6.5, in run D, the maximum ALA
concentration was 66.38 μM (Fig. 2D) whereas in run C where the
pH was 6.5–7.0, it was 40.51 μM (Fig. 2C). Thus, pH control is critical
for ALA production. It was also reported that pH influenced ALA
production by E. coli harboring the R. palustris KUGB306 hemA gene
(40). In a pH range (6.0–6.5), the amount of ALA very slowly
degraded. ALA was significantly enhanced when the pH was
maintained at 6.5 and the degradation rate of ALA increased at high
pH (36). Although extracellular ALA content in runs B and D was
FIG. 3. Time profiles of residual acetic acid (closed circles), butyric acid (closed diamonds), propionic acid (closed squares), succinic acid (closed triangles) and LA (open squares) in
the reactor operated under the conditions as were described in Materials and Methods.
BIOMASS AND ALA PRODUCTION UNDER LIGHT AND DARK CONDITIONS 663
about the same, 2-steps controlling DO and pH increased ALA
production rates and its stability (Fig. 2D). The maximum biomass
of runs C and D were 4.3 and 3.8 gDCW/l, respectively (Figs. 2C and
2D). The reactor was operated under the conditions as run D, but the
precursors and the inhibitor were not added, at 28 h, the maximum
biomass and extracellular ALA were 2.8 gDCW/l and 15.20 μM,
respectively (data not shown).
Runs A and B were operated under different DO levels. The profiles
of VFA, succinate, and LA of run A were rapidly decreased as compared
with run B (Figs. 3A and 3B). After 20 h, the time at which LA was
added, about 50% of VFA was consumed. Accordingly, the growth rate
of the bacterium slightly steadied. After the bacterium adjusted the
metabolic pathways, growth was continued (Figs. 2A and 3A). Two
steps controlling the DO and addition of LA at 16 h accelerated the
consumption of VFA, succinate, and LA (Fig. 3C). As shown in Fig. 3C
and Fig. 3D, lower pH caused slower LA utilization.
It should be noted that diauxic growth did not occur in VFA
medium containing acetic acid, propionic acid, and butyric acid. This
was due to propionic, and butyric acids being degraded to acetic acid,
and the bacterium utilizing acetic acid as a carbon source. VFA,
succinate and LA were completely consumed in all experiments
tested. The investigations indicate that DO affected the bacterial
growth rate and ALA production while pH influenced the ALA stability.
The bacterium KG31 produced ALA the quantity was relatively low.
The levels of ALA to be produced from photosynthetic bacteria were in
range of 0.58–27,500 μM; among these, R. sphaeroides was intensively
studied for ALA production. For example, R. sphaeroides CR-720 grown
in glucose and glycine as the sole sources of carbon and nitrogen
produced 27.5 mM ALA (41). However, R. palustris metabolized acetic
664 CHOORIT ET AL.
acid, propionic acid and butyric acid effectively. Thus, cultivations of the
bacterium in organic wastewater or wastewater after hydrogen/
methane production are of interest in terms of pollution reduction,
and the by-product contained cells and ALA which can be used for
promoting plant growth.
Stability of ALA The stability of ALA and the number of the
bacterium were determined at various pH values (5–8) during storage
for 30 days at 4°C and 30°C. The pH exhibited a negative effect on the
stability of ALA. At pH 5, 6, 7 and 8 ALA storage at 4°C was 31.67%, 38.08%,
45.52% and 60.21% degraded; the degradation rate of ALA was 36.80%,
36.59%, 52.82% and 75.43% at the same storage pH values but another
temperature. At 4°C, the number of cells at pH 5, 6, 7 and 8 were
8.87 × 103, 6.50 × 103, 3.77 × 103 and 2.63 × 103 c.f.u./ml, while at 30°C,
they were 2.0 × 102, 2.13 × 102, 1.90 × 102 and 1.53 × 102 c.f.u./ml,
respectively. Therefore, high pH decreased ALA stability, and high
temperature decreased the number of cells.
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