Advances in Bioscience and Biotechnology, 2013, 4, 919-924 ABB
http://dx.doi.org/10.4236/abb.2013.410120 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Effects of light intensities and photoperiods on growth
and proteolytic activity in purple non-sulfur marine
bacterium, Afifella marina strain ME
(KC205142)
Sujjat Al-Azad*, Tan Kar Soon, Julian Ransangan
Borneo Marine Research Institute, University Malaysia Sabah, Kota Kinabalu, Malaysia
Email: *sujjat@ums.edu.my
Received 1 July 2013; revised 1 August 2013; accepted 2 September 2013
Copyright © 2013 Sujjat Al-Azad et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Afifella marina st rain ME (KC205142), a purple non-
sulfur bacterium was isolated from mangrove habi-
tats of Sabah. The effects of light intensities and pho-
toperiods on proteolytic activity in Afifella marina
strain ME (KC205142) were investigated. Secretion of
proteolytic enzymes in Afifella marina was prelimi-
narily assessed by skim milk agarose media. Subse-
quently, light intensities, such as, dark, 1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500 and 5000 lux were
used to evaluate the effects on proteolytic activity in
Afifella marina strain ME under anaerobic condition.
After that, the effect of photoperiods on proteolytic
activity was monitored under anaerobic light condi-
tion (3000 lux) at 0 h (0L/24D), 6 h (6L/18D), 12 h
(12L/12D), 18 h (18L/6D) and 24 h (24L/0D) of pho-
toperiod. The highest proteolytic activity of 74.67 U
was recorded at 3000 lux illumination light intensity.
The proteolytic activity in bacterium Afifella marina
strain ME was positively associated with the dry cell
weight. The proteolytic activity of 72.67 U in bacte-
rium Afifella marina strain ME at 18 h (18L/6D) pho-
toperiod is not significantly different (p > 0.05) from
proteolytic activity of 74.67 U recorded at continuous
light (24L/0D) condition. Light intensity of 3000 lux,
culture period of 48 h and a photoperiod of 18 h (18L/
6D) were the optimum parameters for proteolytic
activity in bacterium Afifella marina strain ME.
Keywords: Afifella marina; Light Intensity; Photoperiod;
Proteolytic Activity; Purple Non-Sulfur Bacteria
1. INTRODUCTION
Purple non-sulfur bacteria (PNSB) are anoxygenic pho-
tosynthetic bacteria that prefer to live as photohetero-
trophs. These groups of bacteria also inhabit in a wide
range of natural environments such as sediments [1],
coastal seawaters [2], brackish water [3], mud from the
bottom of tidal pool [3] and lakes [4]. PNSB has been
applied in bioremediation of sardine waste water [5],
aquaculture feed additive to enhance growth and survival
of post larvae of tiger shrimp [6], production of bio-fuel
[7], production of antiviral substances [8], production of
enzymes [9], bioremediation of heavy metal from pol-
luted water [10] and production of single cell protein
[11].
PNSB are also known to produce extracellular enzymes
such as proteases, lipases, esterase and alkaline phos-
phatase [12]. Proteases are well known enzymes for their
wide range of application in food industries, detergent
industries, pharmaceutical industries and aquaculture in-
dustries. Purple non-sulfur bacterium, Rubrivivax gelati-
nosus grown in GM media under anaerobic light condi-
tion for 48 h has been reported to produce significant
level of proteolytic activity [13]. Extracellular proteases
within the bacterium extracellular polymeric substances
matrix play an important role in providing nutrients, and
alter extracellular polymeric substances composition as
they respond to different environmental conditions. The
extracellular enzymes contributed to the nutrition of the
bacterium by hydrolyzing large molecular organic com-
pounds to smaller oligomers and monomeric components
which can directly be absorbed by the cells [14].
Studies concluded that culture substrates, pH and tem-
perature are the major factors that influence the bacte-
rium proteolytic activities. PNSB can photoassimilate a
*Corresponding author.
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S. Al-Azad et al. / Advances in Bioscience and Biotechnology 4 (2013) 919-924
920
wide variety of organic compounds such as acetate, py-
ruvate, dicarboxylic acids, fatty acids and sugar [15].
However, high level of complex organic polymers in-
duces the proteolytic activities, while simple compounds
inhibit the bacterium proteolytic activities [16]. Low
temperature always results in slow biological activity
while high temperature around 40˚C has been reported to
inhibit the growth of PNSB [17]. A temperature of 30˚C
was reported to be the optimum temperature for prote-
lytic activities in bacteria such as Bacillus sp. [18]. The
maximal protease production was reported in basal me-
dium, where pH 8 was documented to be the optimum
pH value for proteolytic activity in Bacillus sp. [19].
Light intensity is an important factor that controls the
growth of PNSB. The optimum light intensity for the
growth of PNSB is in the range of 2500 lux to 3000 lux.
The highest dry cell weight of 5.6 g/l of Rhodocyclus
gelatinosus is obtained when cultured in glutamate-
malate (GMM) media at 3000 lux of illumination light
intensity [17]. Azad et al. [6] recorded the highest dry
cell weight of 4.8 g/l in Rhodovulum sulfidopilum that
was grown in GMM media under anaerobic condition at
2500 lux light intensity and a temperature of 30˚C ± 2˚C.
Protease production in Rubrivivax gelatinosus was re-
ported to be light inducible, therefore, it is likely that the
light intensity also influences the proteolytic activity in
PNSB. However, limited information is known about the
effects of light intensities and photoperiods on the prote-
olytic activities in PNSB. Therefore, the objectives of
this study were to assess the effects of light intensities
and photoperiods on the proteolytic activities in Afifella
marina strain ME.
2. MATERIALS AND METHODS
2.1. Bacterium and Culture
Afifella marina, strain ME (KC205142) was obtained
from the culture collection of Borneo Marine Research
Institute, University Malaysia Sabah, Malaysia. This
strain was isolated from exposed mangrove mud sample
of KingFisher, Sabah, Malaysia. A single colony of
Afifella marina strain ME was inoculated in Mc. Corney
bottle containing 25 ml of 112 media and incubated an-
aerobically under 2500 lux illumination light intensity at
a temperature of 30˚C ± 2˚C for 7 days. The composition
of 112 media (g/l) was: 10.0 g of yeast-extract, 0.5 g of
magnesium sulphate, 30 g of sodium chloride and 1.0 g
of dipotassium hydrogen phosphate.
2.2. Inoculum Preparation
One milliliter of the best grown culture was taken from
previously culture bottle and transferred into a new Mc.
Corney bottle containing 24 ml of freshly prepared ster-
ile 112 media. The bottles were then incubated the same
way as stated previously. A 48 h culture was used as the
inoculum in subsequent experiments.
2.3. Preliminary Test for Proteolytic Activity in
Afifella marina Strain ME
Afifella marina strain ME was tested for the production
of proteolytic enzyme by incubating the cells on solidi-
fied 112 media supplemented with 10% skim milk. A 48
h culture plate was opened and observed the present of
clear zone around the bacteria colony.
2.4. Proteolytic Activity Characterization
A 48 h inoculum with 10% (v/v) was inoculated into
triplicate bottles containing sterilised 112 media and in-
cubated anaerobically at a temperature of 30˚C ± 2˚C
under 2500 lux of light intensity for 120 h. Three bottles
from each level of light intensity were selected randomly
after 0 h, 24 h, 48 h, 96 h, and 120 hours of incubation to
analyse the dry cell weight (g/l) and proteolytic activity
(U). After 0 h, 24 h, 48 h, 72 h, 96 h and 120 h, three
bottles of culture were taken randomly for analysis of the
dry cell weight (g/l) and proteolytic activity (U).
2.5. Proteolytic Activity in Afifella marina Strain
ME with Levels of Light Intensities
Proteolytic activities in bacterium Afifella marina strain
ME was monitored with various light intensities. Tripli-
cate bottles of 112 media were inoculated with 10% (v/v)
48 h inoculum and incubated anaerobically at 30˚C ± 2˚C
under dark, 1000, 1500, 2000, 2500 (control), 3000,
3500, 4000, 4500 and 5000 lux illumination of light in-
tensities.
2.6. Proteolytic Activity in Afifella marina Strain
ME with Levels of Photoperiod
Triplicate bottles of 112 media were inoculated with
10% (v/v) 48 h of inoculum and incubated anaerobically
at 30˚C ± 2˚C with 3000 lux light intensity under photo-
periods of 0 h light (0L/24D), 6 h light (6L/18D), 12 h
light (12L/12D), 18 h light (18L/6D) and 24 h light
(24L/0D). The light intensity of 3000 lux was selected
base on previous experiment, which showed the highest
proteolytic activity in Afifella ma rina strain ME. The dry
cell weight (g/l) and proteolytic activity (U) of Afifella
marina strain ME after 0 h, 24 h, 48 h, 96 h, and 120 h of
incubation were analysed.
2.7. Analytical Parameters
Dry Cell Weight
The bacterium cells were collected by centrifuging 10 ml
of culture at 4400 rpm for 20 minutes at 4˚C. The proc-
ess was repeated 3 times, after washing and re-suspended
Copyright © 2013 SciRes. OPEN ACCESS
S. Al-Azad et al. / Advances in Bioscience and Biotechnology 4 (2013) 919-924 921
the cells in distilled water. Subsequently, the dry cell
weight is obtained by drying the cell pallet in oven at a
temperature of 105˚C until constant weight achieved.
The dry cell weight was calculated as the formula given
by Sawada et al. [20]:


Drycell weightg/l
Final weightInitial weight1000
v

where:
Initial weight = plastic centrifuge tube weight (g)
Final weight = plastic centrifuge tube weight (g) + dry
cell weight (g)
v = Volume of sample (ml)
2.8. Proteolytic Activity
Proteolytic activity in bacterium Afifella marina strain
ME was assayed using azocasein as substrates according
to method described by Winkler and Stuckman [21]. In
this assay, digestion of azoproteins with proteolytic en-
zymes at 38˚C results in the formation of coloured com-
ponents soluble in trichloroacetic acid. This coloured
compound has maximum absorption at a wavelength of
440 nm. The proteolytic activity is expressed as enzy-
matic unit (U), where one unit of proteolytic activity is
defined as the amount of enzyme that produces an in-
crease in absorbance of 0.01 under assay conditions. The
absorbance was measured using 100 VIS spectropho-
tometer (Busk Scientific, USA).
2.9. Statistical Analysis
Statistical analyses were performed using the SPSS
Windows Statistical Package (version 19.0). Tests were
judged with significant at p < 0.05 level. All variables
were tested for normality and homogeneity of variances.
Data which satisfy the assumptions of normality and
homogeneity, parametric tests (one-way ANOVA, Inde-
pendent t-test, and Pearson correlation test) were per-
formed.
3. RESULTS AND DISCUSSION
3.1. Proteolytic Enzymes Production in Afifella
marina Strain ME
A clear zone was observed when Afifella marina strain
ME was grown on skim milk plate under anaerobic light
condition for 48 h (Figure 1). Similar observations were
reported in other studies where purple non-sulfur bacteria
such as Rhodopseudomonas palustris grown in Malate
basal medium at 28˚C [22] and Rubrivivax gelatinosus
KDDS1 grown in Glutamate-malate (GM) medium un-
der anaerobic light conditions at light intensity of 3000
lux and a temperature of 35˚C for 48 h [13] exhibited
proteolytic activity. The result of this study shows that
Afifella marina strain ME do secrete of extracellular pro-
tease under anaerobic light conditions.
3.2. Growth and Proteolytic Activity Profile of
Afifella marina Strain ME
Afifella marina strain ME incubated under anaerobic
light condition at light intensity of 2500 lux and at a tem-
perature of 30˚C had shown a sigmoid growth curve. The
growth began with a negligible lag phase, followed by an
exponential phase until 72 h of incubation then entered a
deceleration phase until 120 h of incubation (Figure 2).
The highest dry cell weight of 4.34 g/l was achieved at
72 h culture. The optimum culturing period was similar
to other studies. Rhodomicrobium vannielii grown in GM
media produced the highest dry cell weight of 1.1 g/l at
72 h culture under anaerobic light condition (2000 lux)
[23]. Rhodovulum sulfidophilum grown in sardine proc-
essing effluence recorded highest dry cell weight of 2.8
g/l at 72 h culture under anaerobic light condition (2500
Bacterial colony
Clear zone
Skim milk with agar
Figure 1. Zone clearance by Afifella marina strain ME grown
on skim milk plate under anaerobic light condition (2500 lux)
and at a temperature of 30˚C ± 2˚C for 48 h.
Figure 2. Dry cell weight (g/l) and proteolytic activities (U) of
Afifella marina strain ME in 112 media incubated under an-
aerobic light condition (2.5 k lux illumination, control) for the
period of 120 hours at a temperature of 30˚C ± 2˚C.
Copyright © 2013 SciRes. OPEN ACCESS
S. Al-Azad et al. / Advances in Bioscience and Biotechnology 4 (2013) 919-924
922
lux) [5]. The decrease in dry cell weight after 72 h was
believed to be due to the cells had entered stationary
phase or were dead [23]. This suggested the optimum dry
cell weight production of Afifella marina strain ME is at
72 h culture.
Interestingly, the proteolytic activity corresponding
with growth where the proteolytic activity of Afifella
marina strain ME was increased drastically to 54.67 U
within 48 h of incubation. Subsequently, the level prote-
olytic activity of Afifella marina remains constant at 54.7
U until 120 h of incubation. The maximum proteolytic
activity of 54.7 U was recorded at 48 h of incubation in
this study. The optimum incubation time of 48 h re-
corded in Afifella marina strain ME was in agreement
with Shivanand and Jayaraman [24], where 48 h incuba-
tion was the optimum incubation time for Bacillus aqui-
maris strain VITP4 too. Bacteria were reported to syn-
thesis extracellular enzymes actively within 24 h of in-
cubation. However, the quantitative enzymes activities
might increase until 72 h of incubation [18]. This study
shows that the proteolytic activities of Afifella marina
strain ME optimum at 48 h culture.
The maximum proteolytic activity of 57.4 U at 48 h
culture in Afifella marina strain ME was doubled the
proteolytic activities (25 U) of Rubrivivax gelatinosus
that was grown in GM media under anaerobic light con-
dition (3000 lux) at a temperature of 35˚C [13]. This
suggests that Afifella marina strain ME is a good prote-
olytic enzymes producer among PNSB. Compared to
other microbial proteolytic producers, the proteolytic
activity in Afifella marina strain ME, the purple non-
sulfur bacteria was much lower than that the proteolytic
activity (350 to 410 U) in Bacillus sp. [25]. Maximum
proteolytic activity of Bacillus aq uimaris strain VITP4 of
630 U/ml was recorded when cultured in zobell broth at
37˚C for 48 h of incubation [24]. It is not surprising be-
cause Bacillus spp. is a well known proteolytic enzymes
producer. It is reasonable to believe that higher prote-
olytic activity in Bacillus aquimaris strain VITP4 than
Afifella marina strain ME is due to Bacillus spp. are
naturally biosynthesis higher level of proteolytic en-
zymes than PNSB. However, the proteolytic avtivities in
Afifella marina strain ME (57.4 U) is comparable to pro-
teolytic activity in probiotic bacteria strain TP3B that
isolated from tilapia (51.0 U) [26]. This indicates the
Afifella marina strain ME could be a potential probiotic
bacteria.
3.3. Effect of Light Intensities on Dry Cell
Weight and Proteolytic Activity in Afifella
marina Strain ME
Dry cell weight and proteolytic activity of Afifella ma-
rina strain ME were strongly affected by light intensity.
Increase in light intensities from dark to 3000 lux was
associated with increase in dry cell weight from 1.28 to
4.97 g/l and proteolytic activity from 16.67 ± 2.31 U to
74.67 ± 2.31 U (Figure 3). This observation was com-
parable to other study where the proteolytic activity of
Bacillus aquimaris strain VITP4 grown in zobell broth at
37˚C also nearly corresponded with the growth and it
was maximal at stationary phase [24].
The maximum dry cell weight of 4.97 g/l and prote-
olytic activity of 74.67 ± 2.31 U in Afifella marina strain
ME were recorded at 3000 lux intensity in Afifella ma-
rina strain ME under anaerobic conditions at 48h culture.
These values were significantly higher (p < 0.05) than
that the dry cell weight and proteolytic activities re-
corded at other light intensities. Rhodocyclus gelatinosus
grown in GM media also yield the maximum dry cell
weight of 5.6 g/l under anaerobic light condition at light
intensity of 3000 lux and a temperature of 30˚C ± 2˚C
[17]. PNSB prefers to grow as photoheterotrophs in the
presence of light and dissolved organic matter [3]. There-
fore, increase in dry cell weight is associated with in-
crease in light intensity. On the other hand, proteolytic
activity in purple non-sulfur bacterium, Rubrivivax ge-
latinosus was reported to be light inducible and light
dependent [13]. The light inducible proteinase complex
is associated with thylakoid membranes. The light in-
ducible serine type proteinase is released under light
stress condition [27]. So, higher proteolytic activity of
Afifella marina strain ME in higher light intensity sug-
gests that it may be also light dependent.
However, the dry cell weight and proteolytic activity
were then decreased to 3.2 g/l and 50.00 ± 2.00 U, re-
spectively at 4000 lux of light intensity. Rhodomicrobium
bvanielii grown in GM media under anaerobic light con-
dition at 4000 lux also results in low growth of 1.1 g/l
[23]. This simply indicates that high light intensity of
4000 lux inhibit the growth of cell due to the heat
generated in the medium. The lower the proteolytic
activity at high light intensity of 4000 lux can be
explained by the low dry cell weight. The cells were
Figure 3. Effect of light intensities on maximum dry cell
weight (g/l) and proteolytic activities (U) in Afifella marina
strain ME in 112 media incubated under anaerobic light condi-
tions at a temperature of 30˚C ± 2˚C.
Copyright © 2013 SciRes. OPEN ACCESS
S. Al-Azad et al. / Advances in Bioscience and Biotechnology 4 (2013) 919-924 923
dead due to high temperature in the culture then resulting
in proteolytic activity also decreased.
This study showed a strong positive correlation (R =
0.839, p < 0.01) between proteolytic activity and yield in
dry cell weight. One of the functions of extracellular
enzymes in heterotrophic microorganisms is contributed
to the nutrition by hydrolyzing large molecular organic
compounds before being absorbed by cells [14]. The
higher proteolytic activity at higher dry cell weight sug-
gests that Afifella marina might secrete proteolytic en-
zyme to hydrolyze high molecular weight compound
outside the cell, prior to the uptake of growth. However,
further studies are essential to validate this assumption.
3.4. Effect of Photoperiod on Dry Cell Weight
and Extracellular Proteolytic Activity in
Afifella marina Strain ME
Dry cell weight and proteolytic activity in Afi fella marina
strain ME was influenced by photoperiod where increase
in photoperiod associated with increase in proteolytic
activity (Figure 4). The highest dry cell weight of 4.97
g/l and proteolytic activity of 74.7 ± 2.31 U were re-
corded at continuous light condition (24L/0D). These
values were significantly higher (p < 0.05) than that dry
cell weight and proteolytic activity at dark (0L/24D), 6 h
light (6L/18D) and 12 h light (12L/12D). It is not sur-
prising because continuous light condition favours the
growth of photosynthetic bacterial cells [28].
However, there was no significance difference (p >
0.05) in dry cell weight and proteolytic activity between
continuous light condition (24L/0D) (74.7 ± 2.31 U) and
18 h (18L/6D) light condition (72.7 ± 4.6 U). This indi-
cates that reducing in photoperiod still works without
significantly reducing the growth and production of me-
tabolites is possible. Eroglu et al. [29] reported Rhodo-
bacter sphaeroides grown in olive mill wastewater under
anaerobic light condition (200 W/m2) at a temperature of
32˚C showed no significant difference in dry cell weight
Figure 4. Effect of photoperiod on proteolytic activities (U) in
Afifella marina strain ME in 112 media incubated under an-
aerobic light condition (3000 lux) at 48 h culture and a tem-
perature of 30˚C ± 2˚C.
between 12 h light (12L/12D) of 0.5 g/l and continuous
light condition (24L/0D) of 0.55 g/l. Moreover, Liqin et
al. [30] also reported 18 h photoperiod (18L/6D) was the
optimum photoperiods for extracellular polymeric sub-
stances (105 g/l) produced by photosynthetic bacteria at
a temperature of 30˚C and light intensity of 80 µE/m2s.
Thus, present study suggests that the continuous light
culture condition (24L/0D) can be replaced by 18 h of
photoperiods (18L/6D) to yield the similar proteolytic
activity in Afifella marina strain ME.
4. CONCLUSION
Afifella marina strain ME (KC205142) produces pro-
tease under anaerobic light conditions at temperature of
30˚C ± 2˚C. The proteolytic activity was positively
correlated with the dry cell weight. Optimum proteolytic
activity was recorded at 48 h of incubation. The optimum
light intensity and photoperiods of proteolytic activity in
Afifella marina strain ME are at 3000 lux and 18 h (18L/
6D) light, respectively.
5. ACKNOWLEDGEMENTS
This study was supported by the project FRG0274-SG-2/2010, a grant
from Ministry of Higher Education Malaysia to University Malaysia
Sabah.
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