Effects of light intensities and photoperiods on growth and proteolytic activity in purple non-sulfur marine bacterium, <i>Afifella marina</i> strain ME (KC205142)

doi:10.4236/abb.2013.410120

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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: *[email protected]

Received 1 July 2013; revised 1 August 2013; accepted 2 September 2013

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.

OPEN ACCESS

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

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





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.

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.

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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