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Effects of different light source and media on growth and production of phycobiliprotein from freshwater cyanobacteria
Accepted Manuscript Effects of different light source and media on growth and production of phycobiliprotein from freshwater cyanobacteria Helena Khatoon, Lai Kok Leong, Norazira Abdu Rahman, Sohel Mian, Hasina Begum, Sanjoy Banerjee, Azizah Endut PII: DOI: Reference:
S0960-8524(17)31881-3 https://doi.org/10.1016/j.biortech.2017.10.052 BITE 19093
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
19 July 2017 11 October 2017 12 October 2017
Please cite this article as: Khatoon, H., Kok Leong, L., Abdu Rahman, N., Mian, S., Begum, H., Banerjee, S., Endut, A., Effects of different light source and media on growth and production of phycobiliprotein from freshwater cyanobacteria, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.10.052
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1
EFFECTS OF DIFFERENT LIGHT SOURCE AND MEDIA ON GROWTH AND
2
PRODUCTION
3
CYANOBACTERIA
OF
PHYCOBILIPROTEIN
FROM
FRESHWATER
4 5
Helena Khatoon a*, Lai Kok Leong
b
6
Begum d, Sanjoy Banerjee d, Azizah Endut e
,
Norazira Abdu Rahman b, Sohel Mian
b,c
, Hasina
7 8
a
9
Terengganu, Malaysia
Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala
10
b
11
Kuala Terengganu, Malaysia
12
c
13
Sylhet, Bangladesh
14
d
15
e
16
Abidin University, 21300 Kuala Terengganu, Terengganu, Malaysia
School of Fisheries & Aquaculture Sciences, Universiti Malaysia Terengganu, 21030
Department of Fisheries Biology & Genetics, Sylhet Agricultural University, 3100
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
East Coast of Environmental Research Institute, Gong Badak Campus, Sultan Zainal
17 18 19
Abstract
20 21
The aim of this study was to determine the effect of different light sources and media
22
(wastewater and BBM) on the growth of Pseudanabaena mucicola and its
23
phycobiliprotein production. Results showed that P. mucicola grown in white light
24
using wastewater as medium attributed higher biomass (0.55 g L-1) and when extracted
25
with water, also showed significantly higher (P < 0.05) production (237.01 mg g-1) and
26
purity (1.14) of phycobiliprotein. This study validated that phycobiliprotein extracted
27
from P. mucicola using water can be food grade natural blue pigment. Moreover,
28
cyanobacteria grown in wastewater could cut down the production cost of
29
phycobiliprotein.
30 31
Keywords: Phycobiliprotein, Cyanobacteria, Growth, Wastewater, Light Source
32 33
1. Introduction
34 35
Cyanobacteria (blue-green algae) uphold several structural features resemble to
36
those of bacteria. However, these are classified with algae due to their chlorophyll a and
37
other compounds content. Many cyanobacteria species are potential producers of value-
38
added bioactive compounds such as chlorophyll a, b and c, β-carotene, astaxanthin,
39
xanthophylls and phycobiliprotein (Begum et al., 2016). Moreover, cyanobacteria have
40
gained great attention as a natural antioxidant source because of their bioactive
41
compounds composition. Some cyanobacteria species have the ability to produce
42
bioplastic poly-3-hydroxybutyrate under nitrogen deprivation and photoautotrophy
43
(Taepucharoen et al., 2017). Phycobiliprotein are the major light-harvesting pigments of
44
cyanobacteria, red algae, glaucocystophyte and cryptophyte (Sidler, 1994). They are
45
highly fluorescent because of their covalently bound, linear tetrapyrrole chromophores
46
known as bilins (Paliwal et al., 2017). Phycobiliprotein includes blue colored
47
phycocyanin, bluish green colored allophycocyanin and red colored phycoerythrin
48
(Grossman et al., 1993). In cyanobacteria, phycobiliprotein may comprise up to 40% of
49
its total soluble protein content. It is a high-value algae products used in food and
50
cosmetic industries, nutraceuticals and pharmaceuticals, as well as in biomedical
51
research and clinical diagnostics. Different types of cyanobacteria have been recognized
52
as rich source of bioactive compounds (Abarzua et al. 1999; Dahms et al. 2006). Most
53
of the bioactive compounds isolated from cyanobacteria consist of an amino acid and
54
fatty acid portion. Antibacterial, antifungal, antialgal, antiprotozoan, and antiviral are
55
the secondary metabolites isolated from different cyanobacteria. In addition,
56
cyanobacteria such as Phormidium sp., Spirulina platensis, Lyngbya majuscule and
57
Schizochytrium sp. are source of a variety of compounds such as polysaccharides, lipids,
58
proteins, vitamins, sterols, enzymes, pharmaceuticals and some other well recognized
59
fine chemicals (Sørensen et al., 2013; Borowitzka 1992; Pulz and Gross 2004). It has
60
been using as coloring agent in food industry, specially candies and soft drinks. Due to
61
its non-toxic and non-carcinogenic properties, phycobiliprotein is gaining prominence
62
worldwide as a natural food colorants in replacing the potentially toxic and carcinogenic
63
synthetic food colorants (Pandey et al., 2013). It is also considered as a high value
64
natural dye, of which the native pigment price are US$ 3 to US$ 25 mg-1 and can reach
65
up to US$ 1500 mg-1 for certain cross-linked pigments.
66 67
However, the growth of microalgae and pigment production are affected by many
68
factors such as light irradiances, photoperiods, pH, nutrient limitation, nitrogen
69
supplements and salinity (Hemlata and Fatma, 2009). On the other hand, some species
70
are also capable of producing certain metabolites under extreme culture condition, in
71
order to overcome and adapt to the biotic or abiotic stress conditions (Paliwal et al.,
72
2017). Therefore, the production of metabolites during microalgae growth is important
73
to obtain maximum productivity. Ores et al. (2016) reported that by using an ultrasonic
74
homogenizer, it was possible to obtain high extraction yields of phycobiliproteins from
75
cyanobacteria. In addition, extraction method with drying, freezing and maceration were
76
also effective.
77 78
Light on its own plays a very important role in growth and pigment accumulation of
79
cyanobacteria. Most cyanobacteria have the ability to control the amount of their
80
photosynthetic antenna pigments, phycobiliprotein based on the spectrum of ambient
81
light which is also known as chromatic adaptation (Vogelmann and Scheibe et al.,
82
1978). Upon exposure to changes in light color or high irradiance, cyanobacteria
83
dramatically change its pigmentation composition to optimize their light harvesting
84
mechanism (Grossman et al., 2001; Ojit et al., 2015). The changes in cell pigmentation
85
in response to specific spectral illuminations is due to the moderation of the red colored
86
phycoerythrin and the blue-colored phycocyanin, with a predominance of phycoerythrin
87
in green-light-grown cells and of phycocyanin in red-light grown cells (Paliwal et al.,
88
2017). Thus, even a slight fluctuation in light factors may lead to morphological and
89
physiological changes in cyanobacteria.
90 91
In addition, the high cost of the commercial culture medium is also one of the
92
challenges faced in cyanobacteria production. Generally, cyanobacteria can grow in
93
various aquatic environments such as river, lake and wastewater depending on the
94
nutrient availability specially nitrogen and phosphorus. Aquaculture wastewater from
95
fish and shrimp farming was identified as a potential media for microalgae culture as
96
they ensure ample nitrogen and phosphorus (Khatoon et al., 2016). It can be used as a
97
potential cost effective medium for cyanobacteria culture instead of being released as
98
waste to the environment which may lead to a severe pollution problem.
99 100
There is little data on the growth and phycobiliprotein production of the
101
cyanobacteria Pseudanabaena mucicola, especially under different growth factors such
102
as light and media. In addition, extraction step is very important because of the
103
thickness of the cell wall of the microorganisms and the intracellular location of
104
phycobiliprotein (Ores et al., 2016).
105
There is great demand of phycobiliproteins at commercial level. However, there is a
106
scarcity of information in this field because the quality of the pigments depends on the
107
media and different light source use to culture cyanobacteria. The potential of
108
phycobiliprotein extraction by using chemical free water extraction in comparison to the
109
commonly used phosphate buffer was also determined. In addition, this study also
110
highlighted the potential use of wastewater as an alternative to the expensive
111
commercial medium. Findings can be manipulated and applied for specific uses and for
112
high mass production of P. mucicola commercially.
113 114
2. Materials and Methods
115 116
2.1. Sample collection, culture and maintenance
117
Freshwater cyanobacteria, P. mucicola was obtained from Laboratory of Live
118
Food Culture, Institute of Tropical Aquaculture, University Malaysia Terengganu. The
119
pure cyanobacterial species was cultured in an Erlenmeyer flask containing the Bold
120
Basal Media (BBM) (Stein, 1980). Sub culturing was done every two weeks in order to
121
maintain healthy and good stocks. Then the stock cultures were used during the
122
experiment.
123 124
2.2. Media preparation
125
Two different media (wastewater and Bold Basal medium (BBM)) were
126
prepared. BBM, a commercial media for freshwater algae involves the preparation of
127
stock solutions which consisted of the macronutrients and trace metal solutions. On the
128
other hand, wastewater from cage culture in Kenyir Lake, Malaysia was collected to be
129
used as media. The wastewater was filtered using 0.45µm membrane filter before it was
130
sterilized by UV light (model: RIBAO UV-6 Submersible, Singapore) for 72 hours.
131
Afterthat wastewater was kept at room temperature until further used.
132 133
2.3. Culture of cyanobacteria
134
Experiment was carried out using 2 L flask filled with 1.5 L culture media under
135
three different light sources (white light, blue light, and natural light). The light sources
136
used for culturing cyanobacteria are cool white and blue fluorescent tubes (Philips,
137
USA). Two different culture media, which were BBM and wastewater were used. The
138
pH of the wastewater and BBM media were adjusted 7.5 and was measured using a pH
139
meter (Orion, USA). Culture with white light (180 µmol m-2s-1) and blue light (110
140
µmol m-2s-1) source were maintained inside the culture room at 25 oC and 24 hours
141
photoperiod and was measured using light meter (Quantum Light Meter, Spectrum
142
technologies Inc., USA). Meanwhile, the culture under natural condition was kept
143
outside under shade. The initial stock culture was at 10% of culture volume for each
144
treatment.
145
Growth parameters were measured daily. All experimental cultures were
146
harvested at stationary phase. Cells were harvested at their stationary phase by
147
centrifugation at 8000 rpm for 10 min followed by washing twice with sterilized
148
distilled water. The harvested biomass was dried overnight at 40 °C and kept at -20 oC
149
until further used.
150 151
2.4. Growth parameters analysis
152
Cyanobacterial growth was measured using biomass and optical density.
153
Biomass was measured in terms of chlorophyll a according to APHA (1992). The
154
optical density was determined by using a spectrophotometer (UV-VIS 1601,
155
Shimadzu, Japan) and the wavelength used was 650 nm.
156 157
2.5. Extraction of phycobiliprotein from cyanobacteria
158
Dried cyanobacteria biomass (40 mg) was soaked in 10 ml phosphate buffer (pH
159
7.0; 0.1 M) and distilled water, vortexed and stored at 4 °C for 24h. Phycobiliprotein
160
were extracted by centrifuging at 6000 rpm for 10 min after which the supernatants
161
were collected and absorbance were read at different wave lengths (562nm, 615nm, and
162
652nm) by using phosphate buffer and water as blank. The amount of phycocyanin
163
(PC), phycoerythrin (PE) and allophycocyanin (APC) in the samples were calculated
164
according to Siegelman and Kycia (1978). Meanwhile, total phycocyanin,
165
phycoerythrin and allophycocyanin were calculated according to Silveira et al. (2007).
166 167 168
169 170 171
2.6. Purification of Phycobiliprotein The purity of the phycobiliprotein extract was determined by using spectrophotometer at absorbance ratio of A619/A280 and A656/A280 (Reid et al., 1998).
172 173
2.7. Nutrient analysis wastewater and BBM
174
Total ammonia nitrogen, nitrite nitrogen, and phosphate phosphorous of the
175
culture media were determined before P. mucicola culture and after it was harvested.
176
Nutrients analysis were done according to Parsons et al. (1984).
177
chromatography (882 Compact IC Plus, Metrohm, Switzerland) was used to analysed
178
chemical properties of wastewater.
In addition, ion
179 180
2.8. Statistical Analysis
181
The collected data were analyzed using two way analysis of variance (ANOVA) and
182
the significant differences amongst treatments were determined using Tukey test at 95%
183
confidence interval level. All statistical analysis was done using the SPSS (Statistical
184
Package for the Social Sciences) software.
185 186
3. Results and discussion
187 188
3.1. Growth and biomass yield of P. mucicola
189
The optical density and dry biomass yield of P. mucicola cultured under different
190
light sources and media have been shown in Fig. 1 and Fig. 2. In this study, white light
191
had significantly (P < 0.05) highest growth in terms of optical density compared to blue
192
light and natural light (Fig. 1). The highest optical density was obtained by white light
193
and BBM treatment, followed by white light and wastewater media treatment at 0.731
194
and 0.575 abs, respectively. On the other hand, the chlorophyll a content of P. mucicola
195
reached it peaks on Day 8 with the highest value obtained when P. mucicola was
196
cultured under white light and using wastewater media at 2.10 mg m-2. The highest dry
197
biomass was also produced when P. mucicola was cultured under white light source
198
with no significant difference between BMM and wastewater media at 0.577 g L-1 and
199
0.555 g L-1, respectively. This might caused by the Pseudanabaena sp. not similar with
200
other cyanobacteria which grow better in blue light and low light intensity. Besides, P.
201
mucicola may have slower growth in blue light condition.
202 203
Cyanobacteria are influenced by a variety of environmental factors and they play a
204
significant role in the production and composition of the photosynthetic pigments.
205
Factors like light, temperature, pH and nutrient availability are known to influence the
206
amount of various phycobiliprotein in cyanobacteria (Hemlata and Fatma, 2009; Takano
207
et al., 1995). Cyanobacteria have developed unique physiological and morphological
208
features to respond to changes in environmental parameters for instance, nutrient and
209
light (Tandeau de Marsac and Houmard, 1993). Higher growth in terms of optical
210
density and chlorophyll a content under white light is in accordance with Madhyastha
211
and Vatsala (2007), who reported that white light was preferable for chlorophyll
212
synthesis which leads to higher chlorophyll concentration. The higher light intensity of
213
white light also promotes greater biomass production for P. mucicola in this study. The
214
growth of cyanobacteria was directly proportional to light intensity until saturation level
215
(Ravelonandro et al., 2008). Although other cyanobacterial species such as Spirulina sp.
216
showed higher chlorophyll content under blue light (Chauhan and Pathak, 2010), but in
217
this study, P. mucicola showed the lowest chlorophyll content under natural light
218
(wastewater) compared to other light sources. It was reported by Ojit et al. (2015) that
219
the increase in light availability usually lead to a decrease in chlorophyll a content of
220
cyanobacteria as a strategy for prevention of photo-oxidative damage caused by the
221
production of free radicals. Photoinhibition is the inhibition of photosystem II (PSII)
222
activity under strong light. This occurrence is due to an imbalance between the rate of
223
photodamage to PSII and the rate of the repair of damaged PSII. The mechanism of
224
photoinhibition, strong light induces the production of reactive oxygen species (ROS),
225
which directly inactivates the photochemical reaction center of PSII (Murata et al.,
226
2007).
227 228
Excessive light energy absorbed by cells may lead to the formation of free
229
radicals such as singlet oxygen, which can lead to photo-inhibition by oxidizing the key
230
proteins of the photosynthetic apparatus, including the D1 protein of photosystem II
231
(Inoue et al., 2011). In terms of higher growth and biomass production, culture of P.
232
mucicola under white light by using wastewater media is recommended. This finding is
233
in accordance with the study by Khatoon et al. (2016) who also found that cultivation of
234
marine microalgae in wastewater produced higher biomass than commercial media. The
235
use of wastewater as a low cost media can lead to the reduction in overall P. mucicola
236
production cost, especially during mass culture.
237 238
3.2. Phycobiliprotein production and purity
239
The phycobiliprotein production (allophycocyanin, phycocyanin and phycoerythrin)
240
and total content of phycobiliprotein production (sum of the phycocyanin,
241
allophycocyanin and phycoerythrin contents) by P. mucicola in response to different
242
light sources and media are shown in Fig. 2 and Fig. 3 respectively. Water and
243
phosphate buffer were used to extract the phycobiliprotein from the cyanobacterial
244
biomass. In the present study, the production of phycobiliprotein is affected by the
245
changes in light source and extraction solvents. Significantly higher (P < 0.05) total
246
phycobiliprotein content was found in water extract compared to phosphate buffer for
247
all treatments. The highest total phycobliprotein content (237 mg g-1) was found in
248
water extract of P. mucicola when cultured under white light and using wastewater
249
media (Fig. 3). Higher allophycocyanin production was also obtained in similar culture
250
condition (white light, wastewater media) by extraction using distilled water (0.523
251
mg/ml). Meanwhile, higher phycocyanin production was found under blue light and by
252
using BBM. No significant difference was found between the different extraction
253
solvent. There was also no significant difference in phycoerythrin content among
254
treatment and extraction solvent. In terms of purity ratio of phycocyanin, significantly
255
higher purity ratio (P < 0.05) was found in water extract (0.84) of P. mucicola when
256
cultured under white light by using wastewater media (Fig. 4).
257 258
Phycobliprotein
can
be
divided
into
four
different
classes
which
are
259
allophycocyanin (bluish green), phycocyanin (blue), phycoerythrin (purple) and
260
phycoerythrocyanin (orange) (Sekar and Chandramohan, 2007). In this study,
261
phycobiliprotein production varies when cultured under different light sources.
262 263
Light quality is responsible to change the phycobiliproteins composition and
264
colour through complementary chromatic adaptation. The chromatic acclimation
265
procedure allows the cells of cyanobacteria to change its light absorption characteristics
266
to regulate photosynthesis according to light availability in different environments. As a
267
result of this spectacle, the pigment which absorbs the incident wavelengths of light
268
most strongly becomes predominant (Wang et al., 2007). In the present study, the
269
selected strain was capable of complementary chromatic adaptation by synthesizing two
270
different types of phycobilisomes periphery rod depending upon the available light
271
condition. In addition, phycobiliprotein content was significantly correlated with
272
fluorescent white light indicating its important role in cyanobacteria. The present
273
findings are also supported by the work done by Ojit et al. (2015). In the contemporary
274
findings, it was observed that white light was the suitable light source for the enriched
275
production of phycobiliproteins in the selected cyanobacterial strains investigated.
276
According to Ojit et al. (2015) fluorescent white light was the most suitable light source
277
for the enhanced production of phycobiliproteins in almost all cyanobacterial strains. In
278
addition, their study also investigated that when strains were exposed to red light, their
279
PE content decreased but simultaneously phycocyanin (PC) increased when compared
280
to other light qualities. Similarly it was observed in PC rich cultures i.e. in red light, PC
281
quantity decreased and PE production increased in blue light. The chromatic adaptation
282
ability of cyanobacteria comprises of three complex pathway which includes regulation
283
by a phytochrome class photoreceptor (highly responsive to green and red light), a
284
complex two component signal transduction pathway and also reaction based on the
285
redox state (Paliwal et al., 2017). In the present study, higher amount of phycocyanin
286
was found under blue light compared to white light and natural light. Its concentration
287
(0.419 mg mL-1) is also higher compared to the phycocyanin obtained in a study by
288
Ores et al. (2016) on Spirulina sp. LEB 18 (120 µg mL-1). According to Lönneborg et
289
al (1985), phycocyanin content in cyanobacterial cells increase with low light intensity
290
due to the chromatic adaptation ability. Thus, the lower phycocyanin in natural light
291
may be due to the higher light intensity compared to other light sources. Culture under
292
natural condition was exposed to high light intensities (0.02 – 700 µmol m-2 s-1 ) and
293
12:12 h light dark period. According to Raps et al. (1983), with an increase in light
294
intensity, the concentrations of chlorophyll a and phycocyanin decrease. At higher light
295
intensities, the cells lessen the amount of thylakoid membranes and phycobilisomes to
296
decrease the quantity of radiant energy absorbed as possible (Tandeau de Marsac and
297
Houmard, 1993). Another study by Lee et al. (2017) also found lower phycocyanin
298
production when cyanobacterial, Nostoc sp. NK was cultured under white light
299
compared to red and plant fluorescent light. Whereas, similar with the study by Cuellar-
300
Bermudez et al. (2014), phycoerythrin has been found to occur in much lower
301
concentration compared to other pigments because it is mainly affected by changes in
302
pH rather than light.
303 304
Different pH of the medium also affects the phycobiliprotein by changing the
305
culture colour of cyanobacteria. The productions of phycobiliproteins were greatly
306
affected by the pH which might indicate the inability of the cyanobacteria to maintain a
307
constant internal pH. At lower pH 7.5 Anabaena sp. NCCU-9, resulted in reduced
308
phycobiliproteins extraction. This may be due to the extreme buffer’s pH which might
309
cause internal electrostatic attraction by changing the charge on protein giving net
310
positive charge and at this stage protein open up and bound solvent is lost resulting in
311
the denaturation of protein (Roe, 2000).
312
313
Phosphate buffer is expensive, preparation is time consuming and poor purity of
314
the selected cyanobacteria in their final targeted product. Therefore, water is evaluated
315
in this study, as an alternative solvent to extract phycobiliproteins from selected
316
cyanobacteria. In the present study water was used as a solvent to extract
317
phycobiliproteins. It was shown that water solvent had the best extractive performance,
318
not only due to their capacity to extract higher quantity of proteins, but also it
319
circumvents the extraction of chlorophylls. Similarly, Martins et al. (2016) reported
320
that water extract significantly increased the amount of phycobiliproteins when
321
compared
322
conformational structure or chromophore structural integrity.
with
the
conventional
methodology,
without
compromising
their
323 324
The extraction solvent used also affects the allophycocyanin, phycocyanin and
325
phycoerythrin content. The selection of extraction solvent that can lyse the cell wall to
326
increase the seepage of phycobiliprotein pigment is important. In this study,
327
allophycocyanin, one of the pigments production of P. mucicola was significantly
328
higher in all treatments when water was used as the extraction solvent. Whereas, when
329
the cyanobacteria was extracted using phosphate buffer, the most abundant pigment
330
found under phycobiliprotein is the phycocyanin (blue), compared to phycoerythrin and
331
allophycocyanin which is similar to the study by Walter et al. (2011). Hemlata et al.
332
(2011) also reported better phycocyanin and total phycobiliprotein extraction when
333
using phosphate buffer, although with much lower value compared to this study. In
334
terms of total phycobiliprotein production, higher production was obtained in all
335
treatment when P. mucicola was extracted with water, compared to phosphate buffer.
336
This finding is crucial, especially if the extracted phycobiliprotein is intended for human
337
use as it is chemical free.
338 339
In general, the high allophycocyanin, phycocyanin and phycoeryhtrin content
340
and total phycobiliprotein found in P. mucicola (water extract) when cultured in
341
wastewater media might lead to the reduction in the overall cost, especially during mass
342
scale production for commercial purposes. The culture condition, especially nitrogen
343
and carbon sources in the form of culture media, also determines the production of
344
phycobiliprotein by cyanobacteria (Seker and Chandramohan, 2008). In general,
345
cyanobacteria requires nitrogen sources for growth. Cyanobacteria assimilates
346
ammonium ions (NH4+) from external source via an active transport system, while the
347
unprotonated form (NH3) was absorbed by diffusion and is trapped by protonation.
348
Nitrate is taken up by cells via an active transport system before it was reduced to nitrite
349
and then to ammonium (Lioteberg et al., 1996). Under nitrogen depletion, cyanobacteria
350
use phycobiliprotein as a nitrogen source, which can contribute to a decrease in
351
phycobiliprotein production by nblA gene expression (Erikson, 2008). Thus, nitrogen
352
availability plays a very important role in regulating phycobiliprotein and phycocyanin.
353
Eventhough wastewater contain lower amount of nitrogen (TAN and N-NO2), there
354
were no significant decrease in growth and phycobiliprotein content when compared to
355
P. mucicola cultured in BBM. Thus, the wastewater media is capable of providing
356
sufficient amount of nutrient for the cyanobacteria. Collier and Grossman (1994)
357
reported that, deprivation of phosphorus or sulphur can also led to partial or complete
358
decrease of phycobiliprotein by inducing the expression of nblA gene and
359
phycobiliprotein degradation, not only nitrogen.
360
Among the three main pigments of phycobiliprotein, phycocyanin is the most
361
valuable natural blue pigment used in food and pharmaceutical industry due to their
362
colour, fluorescence and antioxidant properties (Kumar et al., 2014). The purity ratio of
363
phycocyanin plays a significant role in commercial applications where it is often
364
evaluated by using absorbance readings. According to Patil et al. (2006), phycocyanin
365
with purity greater than 0.7 is considered as food grade, at ratio 3.9 is considered as
366
reactive grade and above 4.0 as analytical grade. In the present study, higher purity ratio
367
was found in water extract (0.84) of P. mucicola cultured under white light by using
368
wastewater media. The purity ratio was higher than the maximum purity ratio of
369
Spirulina reported by Prabuthas et al. (2011) at only 0.62 purity ratio.
370 371
3.3. Nutrient analysis of wastewater and culture media
372
Wastewater contained sufficient amount of ammonium (6.12±0.01), nitrite (3.80
373
± 0.03), nitrate (3.47 ± 0.02), phosphate (7.20 ± 0.03), calcium (5.40 ± 0.12), potassium
374
(5.45 ± 0.01), sodium (14.25 ± 0.01) and sulphate (5.05 ± 0.02) for culture of
375
microalgae. Physical parameters such as pH (8.25± 0.02), temperature (26.01 ± 0.01)
376
and dissolved oxygen (5.50 ± 0.02) were in the recommended range suitable for
377
cyanobacteria culture.
378 379
Cyanobacteria growth was dependent on a suitable supply of essential
380
macronutrients (nitrogen and phosphorus) but excess nutrients may have toxic effects
381
on the cell metabolism. Excessive nutrients content in commercial media may perhaps
382
affected the selected cyanobacteria growth in the present study. On the other hand,
383
nitrogen and phosphorous are the major nutrients required to grow microalgae. In this
384
study, aquaculture wastewater ensured sufficient amount of nitrogen and phosphorous
385
for proper growth of cyanobacteria. This would facilitate the removal of nutrients from
386
wastewater and production of microalgae biomass concurrently Abdulsada (2014).
387 388
Table 1 showed the nutrient analysis of wastewater before P. mucicola culture and
389
after P. mucicola was harvested. In the present study, both P. mucicola culture by using
390
BBM and wastewater showed a decrease in the nitrite, phosphorus and ammonia
391
content after the culture were harvested. When cultured using wastewater, the highest
392
TAN and N-NO2 uptake of P. mucicola was under white light treatment at 0.142 mg L-1
393
and 1.539 mg L-1, respectively. Initially, the TAN and N-NO2 content of wastewater
394
media was at 6.32 ± 0.01 mg L-1 and 6.50 ± 0.01 mg L-1, respectively. Meanwhile, when
395
P. mucicola was cultured using BBM media, the highest TAN uptake was observed
396
under natural light and N-NO2 uptake was higher under white light at 0.205 mg L-1 and
397
5.424 mg/L, respectively. The initial TAN and N-NO2 of BBM was at 0.483 mg L-1 and
398
12.990 mg L-1. No significant difference (P > 0.05) was found in the P-PO4 content
399
before and after culture of P. mucicola under all treatments.
400 401
Cyanobacteria have developed highly and efficient uptake and retention
402
mechanisms for three nutrient anions: bicarbonate, nitrite, and phosphate (Badger and
403
Price, 2003). P. mucicola culture under white light treatment and natural light treatment
404
utilized most nitrite in both waste water and BBM treatments. However, in blue light
405
treatment, the high nitrite and phosphorus level after harvest in both waste water and
406
BBM may be caused by the slow growth and nutrient uptake by the P. mucicola cells.
407
According to Hu et al. (2000), the nitrate uptake rate increased proportionally with
408
increasing light intensity or parallels to photosynthetic activity. In this study, blue light
409
have lower light intensity compared to white and natural light. Thus, the culture of P.
410
mucicola by using wastewater under white light treatment may serve as an effective
411
way to treat and reuse wastewater.
412 413
Conclusions
414
The growth, biomass yield and phycobiliprotein production as well as purity of
415
P. mucicola varied when cultured under different light sources and media. High growth,
416
chlorophyll a concentration, biomass and phycobiliprotein production were found when
417
P. mucicola was cultivated using wastewater under white light source. Moreover, it was
418
found that extraction of phycobiliprotein by using water, which is chemical free,
419
produced higher phycobiliprotein concentration compared to phosphate buffer. Further
420
studies are warranted for the confirmation and validation of other environmental factors
421
suitable for the production of phycobiliproteins in cyanobacteria.
422 423
Acknowledgement
424 425
This study was supported by the Ministry of Higher Education, Malaysia, through
426
Fundamental
Research
Grant
427
FRGS/1/2015/WAB01/UMT/03/6.
Scheme
(FRGS)
project
No.
428 429
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V., Soccol, C.R., 2011. Study of phycocyanin production from Spirulina
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568
569
Figure captions
570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602
Figure 1. Optical density (abs) versus culture period (day) for P. mucicola cultured under different media (wastewater (WW) and BBM) and light sources (white light (WL), blue light (BL), natural light (NL)). Values are mean ± standard error. Figure 2. Phycobiliprotein pigment production (mg mL-1) of P. mucicola cultured under different media, light sources and extraction solvents.. Values are mean ± standard error. Figure 3. Total phycobiliprotein production (mg mL-1) of P. mucicola cultured under different media, light sources and extraction solvents. Values are mean ± standard error. Figure 4. Purity of phycocyanin from cyanobacteria cultured under different media, light sources and extraction solvents. Values are mean ± standard error.
Optical density (abs)
0.8
BBM WL
0.7
BBM BL
0.6
BBM NL WW WL
0.5
WW BL 0.4
WW NL
0.3 0.2 0.1 0 0
603 604 605
Fig. 1.
1
2
3 4 Culture period (Day)
5
6
7
606 607 0.600
Pigment production (mg mL-1)
BBM WL 0.500
BBM BL BBM NL
0.400
WW WL WW BL
0.300
WW NL 0.200 0.100 0.000 Phosphate buffer
Phycocyanin (PC)
608 609 610 611 612 613 614 615 616 617 618 619 620
Distilled water
Fig. 2.
Phosphate buffer
Distilled water
Allophycocyanin (APC)
Phosphate buffer
Distilled water
Phycoerythrin (PE)
621 622 623 624 625
Total phycobiliprotein (mg G-1)
626 Phosphate buffer
300 250 200 150
100 50 0
WL
BL BBM
627 628 629 630 631 632 633 634 635 636 637 638
Fig. 3.
Water
NL
WL
BL WW
NL
639 640 641 642 643 644 645 646 647 648 1.4
Phosphate buffer
Water
1.2
Purity
1.0 0.8 0.6 0.4 0.2 0.0 WL
BBM
649 650 651 652
BL
Fig. 4.
NL
WL
BL WW
NL
653
Table captions
654
Table 1
655
Nutrient analysis of wastewater before P. mucicola culture and after P. mucicola
656
was harvested. Values are means ± standard errors (n=3).
Culture
Nitrite nitrogen
Phosphate
nitrogen (TAN)
(NO2-N)
phosphorous (P-PO4)
(mg L-1)
(mg L-1)
(mg L-1)
Light
Sample media
Total ammonium
source
Before
Wastewater
-
6.32 ± 0.01
6.50 ± 0.01
5.53 ± 0.06
culture
BMM
-
0.48 ± 0.02
13.09 ± 0.01
11.06 ± 0.03
WL
0.04 ± 0.01
1.53 ± 0.04
4.00 ± 0.01
BL
0.06 ± 0.03
3.25 ± 0.01
4.40 ± 0.01
NL
0.07 ± 0.01
1.32 ± 0.03
5.00 ± 0.08
WL
0.44 ± 0.01
5.42 ± 0.01
10.50 ± 0.01
BL
0.38 ± 0.00
11.64 ± 0.02
10.01 ± 0.02
NL
0.20 ± 0.03
8.10 ± 0.01
9. 80 ± 0.05
Wastewater After harvest
BBM
657 658 659
660 661
662 663
Optical density of cyanobacteria under different light sources and media
Chlorophyll a of cyanobacteria under different light sources and media
Biomass of cyanobacteria under different light sources and media
664 665
Biomass of cyanobacteria was harvest at stationary phase & freeze dried
666 667 668 669
Dried biomass of cyanobacteria was extracted with water and phosphate buffer for phycobiliprotein production
670 671
672 Phycobiliprotein pigment production of cyanobacteria cultured under different media, light sources and extraction solvents
Total phycobiliprotein production of cyanobacteria cultured under different media, light sources and extraction solvents
Purity of phycocyanin from cyanobacteria cultured under different media, light sources and extraction solvents solvents
673
Research highlights
674 675 676 677 678 679 680 681 682 683 684 685 686 687 688
Effect of different light sources and media on the growth of cyanobacteria was determind Effect of different light sources and media on the phycobiliprotein production of cyanobacteria Pseudanabaena mucicola grown in wastewater under white light had significantly higher biomass Cyanobacteria biomass extracted with water had higher production and purity of phycocyanin. Phycobiliprotein extracted using water from P. mucicola can be food grade natural blue pigment. Cyanobacteria grown in wastewater could cut down the production cost of phycobiliprotein.
S0960-8524(17)31881-3 https://doi.org/10.1016/j.biortech.2017.10.052 BITE 19093
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
19 July 2017 11 October 2017 12 October 2017
Please cite this article as: Khatoon, H., Kok Leong, L., Abdu Rahman, N., Mian, S., Begum, H., Banerjee, S., Endut, A., Effects of different light source and media on growth and production of phycobiliprotein from freshwater cyanobacteria, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.10.052
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1
EFFECTS OF DIFFERENT LIGHT SOURCE AND MEDIA ON GROWTH AND
2
PRODUCTION
3
CYANOBACTERIA
OF
PHYCOBILIPROTEIN
FROM
FRESHWATER
4 5
Helena Khatoon a*, Lai Kok Leong
b
6
Begum d, Sanjoy Banerjee d, Azizah Endut e
,
Norazira Abdu Rahman b, Sohel Mian
b,c
, Hasina
7 8
a
9
Terengganu, Malaysia
Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala
10
b
11
Kuala Terengganu, Malaysia
12
c
13
Sylhet, Bangladesh
14
d
15
e
16
Abidin University, 21300 Kuala Terengganu, Terengganu, Malaysia
School of Fisheries & Aquaculture Sciences, Universiti Malaysia Terengganu, 21030
Department of Fisheries Biology & Genetics, Sylhet Agricultural University, 3100
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
East Coast of Environmental Research Institute, Gong Badak Campus, Sultan Zainal
17 18 19
Abstract
20 21
The aim of this study was to determine the effect of different light sources and media
22
(wastewater and BBM) on the growth of Pseudanabaena mucicola and its
23
phycobiliprotein production. Results showed that P. mucicola grown in white light
24
using wastewater as medium attributed higher biomass (0.55 g L-1) and when extracted
25
with water, also showed significantly higher (P < 0.05) production (237.01 mg g-1) and
26
purity (1.14) of phycobiliprotein. This study validated that phycobiliprotein extracted
27
from P. mucicola using water can be food grade natural blue pigment. Moreover,
28
cyanobacteria grown in wastewater could cut down the production cost of
29
phycobiliprotein.
30 31
Keywords: Phycobiliprotein, Cyanobacteria, Growth, Wastewater, Light Source
32 33
1. Introduction
34 35
Cyanobacteria (blue-green algae) uphold several structural features resemble to
36
those of bacteria. However, these are classified with algae due to their chlorophyll a and
37
other compounds content. Many cyanobacteria species are potential producers of value-
38
added bioactive compounds such as chlorophyll a, b and c, β-carotene, astaxanthin,
39
xanthophylls and phycobiliprotein (Begum et al., 2016). Moreover, cyanobacteria have
40
gained great attention as a natural antioxidant source because of their bioactive
41
compounds composition. Some cyanobacteria species have the ability to produce
42
bioplastic poly-3-hydroxybutyrate under nitrogen deprivation and photoautotrophy
43
(Taepucharoen et al., 2017). Phycobiliprotein are the major light-harvesting pigments of
44
cyanobacteria, red algae, glaucocystophyte and cryptophyte (Sidler, 1994). They are
45
highly fluorescent because of their covalently bound, linear tetrapyrrole chromophores
46
known as bilins (Paliwal et al., 2017). Phycobiliprotein includes blue colored
47
phycocyanin, bluish green colored allophycocyanin and red colored phycoerythrin
48
(Grossman et al., 1993). In cyanobacteria, phycobiliprotein may comprise up to 40% of
49
its total soluble protein content. It is a high-value algae products used in food and
50
cosmetic industries, nutraceuticals and pharmaceuticals, as well as in biomedical
51
research and clinical diagnostics. Different types of cyanobacteria have been recognized
52
as rich source of bioactive compounds (Abarzua et al. 1999; Dahms et al. 2006). Most
53
of the bioactive compounds isolated from cyanobacteria consist of an amino acid and
54
fatty acid portion. Antibacterial, antifungal, antialgal, antiprotozoan, and antiviral are
55
the secondary metabolites isolated from different cyanobacteria. In addition,
56
cyanobacteria such as Phormidium sp., Spirulina platensis, Lyngbya majuscule and
57
Schizochytrium sp. are source of a variety of compounds such as polysaccharides, lipids,
58
proteins, vitamins, sterols, enzymes, pharmaceuticals and some other well recognized
59
fine chemicals (Sørensen et al., 2013; Borowitzka 1992; Pulz and Gross 2004). It has
60
been using as coloring agent in food industry, specially candies and soft drinks. Due to
61
its non-toxic and non-carcinogenic properties, phycobiliprotein is gaining prominence
62
worldwide as a natural food colorants in replacing the potentially toxic and carcinogenic
63
synthetic food colorants (Pandey et al., 2013). It is also considered as a high value
64
natural dye, of which the native pigment price are US$ 3 to US$ 25 mg-1 and can reach
65
up to US$ 1500 mg-1 for certain cross-linked pigments.
66 67
However, the growth of microalgae and pigment production are affected by many
68
factors such as light irradiances, photoperiods, pH, nutrient limitation, nitrogen
69
supplements and salinity (Hemlata and Fatma, 2009). On the other hand, some species
70
are also capable of producing certain metabolites under extreme culture condition, in
71
order to overcome and adapt to the biotic or abiotic stress conditions (Paliwal et al.,
72
2017). Therefore, the production of metabolites during microalgae growth is important
73
to obtain maximum productivity. Ores et al. (2016) reported that by using an ultrasonic
74
homogenizer, it was possible to obtain high extraction yields of phycobiliproteins from
75
cyanobacteria. In addition, extraction method with drying, freezing and maceration were
76
also effective.
77 78
Light on its own plays a very important role in growth and pigment accumulation of
79
cyanobacteria. Most cyanobacteria have the ability to control the amount of their
80
photosynthetic antenna pigments, phycobiliprotein based on the spectrum of ambient
81
light which is also known as chromatic adaptation (Vogelmann and Scheibe et al.,
82
1978). Upon exposure to changes in light color or high irradiance, cyanobacteria
83
dramatically change its pigmentation composition to optimize their light harvesting
84
mechanism (Grossman et al., 2001; Ojit et al., 2015). The changes in cell pigmentation
85
in response to specific spectral illuminations is due to the moderation of the red colored
86
phycoerythrin and the blue-colored phycocyanin, with a predominance of phycoerythrin
87
in green-light-grown cells and of phycocyanin in red-light grown cells (Paliwal et al.,
88
2017). Thus, even a slight fluctuation in light factors may lead to morphological and
89
physiological changes in cyanobacteria.
90 91
In addition, the high cost of the commercial culture medium is also one of the
92
challenges faced in cyanobacteria production. Generally, cyanobacteria can grow in
93
various aquatic environments such as river, lake and wastewater depending on the
94
nutrient availability specially nitrogen and phosphorus. Aquaculture wastewater from
95
fish and shrimp farming was identified as a potential media for microalgae culture as
96
they ensure ample nitrogen and phosphorus (Khatoon et al., 2016). It can be used as a
97
potential cost effective medium for cyanobacteria culture instead of being released as
98
waste to the environment which may lead to a severe pollution problem.
99 100
There is little data on the growth and phycobiliprotein production of the
101
cyanobacteria Pseudanabaena mucicola, especially under different growth factors such
102
as light and media. In addition, extraction step is very important because of the
103
thickness of the cell wall of the microorganisms and the intracellular location of
104
phycobiliprotein (Ores et al., 2016).
105
There is great demand of phycobiliproteins at commercial level. However, there is a
106
scarcity of information in this field because the quality of the pigments depends on the
107
media and different light source use to culture cyanobacteria. The potential of
108
phycobiliprotein extraction by using chemical free water extraction in comparison to the
109
commonly used phosphate buffer was also determined. In addition, this study also
110
highlighted the potential use of wastewater as an alternative to the expensive
111
commercial medium. Findings can be manipulated and applied for specific uses and for
112
high mass production of P. mucicola commercially.
113 114
2. Materials and Methods
115 116
2.1. Sample collection, culture and maintenance
117
Freshwater cyanobacteria, P. mucicola was obtained from Laboratory of Live
118
Food Culture, Institute of Tropical Aquaculture, University Malaysia Terengganu. The
119
pure cyanobacterial species was cultured in an Erlenmeyer flask containing the Bold
120
Basal Media (BBM) (Stein, 1980). Sub culturing was done every two weeks in order to
121
maintain healthy and good stocks. Then the stock cultures were used during the
122
experiment.
123 124
2.2. Media preparation
125
Two different media (wastewater and Bold Basal medium (BBM)) were
126
prepared. BBM, a commercial media for freshwater algae involves the preparation of
127
stock solutions which consisted of the macronutrients and trace metal solutions. On the
128
other hand, wastewater from cage culture in Kenyir Lake, Malaysia was collected to be
129
used as media. The wastewater was filtered using 0.45µm membrane filter before it was
130
sterilized by UV light (model: RIBAO UV-6 Submersible, Singapore) for 72 hours.
131
Afterthat wastewater was kept at room temperature until further used.
132 133
2.3. Culture of cyanobacteria
134
Experiment was carried out using 2 L flask filled with 1.5 L culture media under
135
three different light sources (white light, blue light, and natural light). The light sources
136
used for culturing cyanobacteria are cool white and blue fluorescent tubes (Philips,
137
USA). Two different culture media, which were BBM and wastewater were used. The
138
pH of the wastewater and BBM media were adjusted 7.5 and was measured using a pH
139
meter (Orion, USA). Culture with white light (180 µmol m-2s-1) and blue light (110
140
µmol m-2s-1) source were maintained inside the culture room at 25 oC and 24 hours
141
photoperiod and was measured using light meter (Quantum Light Meter, Spectrum
142
technologies Inc., USA). Meanwhile, the culture under natural condition was kept
143
outside under shade. The initial stock culture was at 10% of culture volume for each
144
treatment.
145
Growth parameters were measured daily. All experimental cultures were
146
harvested at stationary phase. Cells were harvested at their stationary phase by
147
centrifugation at 8000 rpm for 10 min followed by washing twice with sterilized
148
distilled water. The harvested biomass was dried overnight at 40 °C and kept at -20 oC
149
until further used.
150 151
2.4. Growth parameters analysis
152
Cyanobacterial growth was measured using biomass and optical density.
153
Biomass was measured in terms of chlorophyll a according to APHA (1992). The
154
optical density was determined by using a spectrophotometer (UV-VIS 1601,
155
Shimadzu, Japan) and the wavelength used was 650 nm.
156 157
2.5. Extraction of phycobiliprotein from cyanobacteria
158
Dried cyanobacteria biomass (40 mg) was soaked in 10 ml phosphate buffer (pH
159
7.0; 0.1 M) and distilled water, vortexed and stored at 4 °C for 24h. Phycobiliprotein
160
were extracted by centrifuging at 6000 rpm for 10 min after which the supernatants
161
were collected and absorbance were read at different wave lengths (562nm, 615nm, and
162
652nm) by using phosphate buffer and water as blank. The amount of phycocyanin
163
(PC), phycoerythrin (PE) and allophycocyanin (APC) in the samples were calculated
164
according to Siegelman and Kycia (1978). Meanwhile, total phycocyanin,
165
phycoerythrin and allophycocyanin were calculated according to Silveira et al. (2007).
166 167 168
169 170 171
2.6. Purification of Phycobiliprotein The purity of the phycobiliprotein extract was determined by using spectrophotometer at absorbance ratio of A619/A280 and A656/A280 (Reid et al., 1998).
172 173
2.7. Nutrient analysis wastewater and BBM
174
Total ammonia nitrogen, nitrite nitrogen, and phosphate phosphorous of the
175
culture media were determined before P. mucicola culture and after it was harvested.
176
Nutrients analysis were done according to Parsons et al. (1984).
177
chromatography (882 Compact IC Plus, Metrohm, Switzerland) was used to analysed
178
chemical properties of wastewater.
In addition, ion
179 180
2.8. Statistical Analysis
181
The collected data were analyzed using two way analysis of variance (ANOVA) and
182
the significant differences amongst treatments were determined using Tukey test at 95%
183
confidence interval level. All statistical analysis was done using the SPSS (Statistical
184
Package for the Social Sciences) software.
185 186
3. Results and discussion
187 188
3.1. Growth and biomass yield of P. mucicola
189
The optical density and dry biomass yield of P. mucicola cultured under different
190
light sources and media have been shown in Fig. 1 and Fig. 2. In this study, white light
191
had significantly (P < 0.05) highest growth in terms of optical density compared to blue
192
light and natural light (Fig. 1). The highest optical density was obtained by white light
193
and BBM treatment, followed by white light and wastewater media treatment at 0.731
194
and 0.575 abs, respectively. On the other hand, the chlorophyll a content of P. mucicola
195
reached it peaks on Day 8 with the highest value obtained when P. mucicola was
196
cultured under white light and using wastewater media at 2.10 mg m-2. The highest dry
197
biomass was also produced when P. mucicola was cultured under white light source
198
with no significant difference between BMM and wastewater media at 0.577 g L-1 and
199
0.555 g L-1, respectively. This might caused by the Pseudanabaena sp. not similar with
200
other cyanobacteria which grow better in blue light and low light intensity. Besides, P.
201
mucicola may have slower growth in blue light condition.
202 203
Cyanobacteria are influenced by a variety of environmental factors and they play a
204
significant role in the production and composition of the photosynthetic pigments.
205
Factors like light, temperature, pH and nutrient availability are known to influence the
206
amount of various phycobiliprotein in cyanobacteria (Hemlata and Fatma, 2009; Takano
207
et al., 1995). Cyanobacteria have developed unique physiological and morphological
208
features to respond to changes in environmental parameters for instance, nutrient and
209
light (Tandeau de Marsac and Houmard, 1993). Higher growth in terms of optical
210
density and chlorophyll a content under white light is in accordance with Madhyastha
211
and Vatsala (2007), who reported that white light was preferable for chlorophyll
212
synthesis which leads to higher chlorophyll concentration. The higher light intensity of
213
white light also promotes greater biomass production for P. mucicola in this study. The
214
growth of cyanobacteria was directly proportional to light intensity until saturation level
215
(Ravelonandro et al., 2008). Although other cyanobacterial species such as Spirulina sp.
216
showed higher chlorophyll content under blue light (Chauhan and Pathak, 2010), but in
217
this study, P. mucicola showed the lowest chlorophyll content under natural light
218
(wastewater) compared to other light sources. It was reported by Ojit et al. (2015) that
219
the increase in light availability usually lead to a decrease in chlorophyll a content of
220
cyanobacteria as a strategy for prevention of photo-oxidative damage caused by the
221
production of free radicals. Photoinhibition is the inhibition of photosystem II (PSII)
222
activity under strong light. This occurrence is due to an imbalance between the rate of
223
photodamage to PSII and the rate of the repair of damaged PSII. The mechanism of
224
photoinhibition, strong light induces the production of reactive oxygen species (ROS),
225
which directly inactivates the photochemical reaction center of PSII (Murata et al.,
226
2007).
227 228
Excessive light energy absorbed by cells may lead to the formation of free
229
radicals such as singlet oxygen, which can lead to photo-inhibition by oxidizing the key
230
proteins of the photosynthetic apparatus, including the D1 protein of photosystem II
231
(Inoue et al., 2011). In terms of higher growth and biomass production, culture of P.
232
mucicola under white light by using wastewater media is recommended. This finding is
233
in accordance with the study by Khatoon et al. (2016) who also found that cultivation of
234
marine microalgae in wastewater produced higher biomass than commercial media. The
235
use of wastewater as a low cost media can lead to the reduction in overall P. mucicola
236
production cost, especially during mass culture.
237 238
3.2. Phycobiliprotein production and purity
239
The phycobiliprotein production (allophycocyanin, phycocyanin and phycoerythrin)
240
and total content of phycobiliprotein production (sum of the phycocyanin,
241
allophycocyanin and phycoerythrin contents) by P. mucicola in response to different
242
light sources and media are shown in Fig. 2 and Fig. 3 respectively. Water and
243
phosphate buffer were used to extract the phycobiliprotein from the cyanobacterial
244
biomass. In the present study, the production of phycobiliprotein is affected by the
245
changes in light source and extraction solvents. Significantly higher (P < 0.05) total
246
phycobiliprotein content was found in water extract compared to phosphate buffer for
247
all treatments. The highest total phycobliprotein content (237 mg g-1) was found in
248
water extract of P. mucicola when cultured under white light and using wastewater
249
media (Fig. 3). Higher allophycocyanin production was also obtained in similar culture
250
condition (white light, wastewater media) by extraction using distilled water (0.523
251
mg/ml). Meanwhile, higher phycocyanin production was found under blue light and by
252
using BBM. No significant difference was found between the different extraction
253
solvent. There was also no significant difference in phycoerythrin content among
254
treatment and extraction solvent. In terms of purity ratio of phycocyanin, significantly
255
higher purity ratio (P < 0.05) was found in water extract (0.84) of P. mucicola when
256
cultured under white light by using wastewater media (Fig. 4).
257 258
Phycobliprotein
can
be
divided
into
four
different
classes
which
are
259
allophycocyanin (bluish green), phycocyanin (blue), phycoerythrin (purple) and
260
phycoerythrocyanin (orange) (Sekar and Chandramohan, 2007). In this study,
261
phycobiliprotein production varies when cultured under different light sources.
262 263
Light quality is responsible to change the phycobiliproteins composition and
264
colour through complementary chromatic adaptation. The chromatic acclimation
265
procedure allows the cells of cyanobacteria to change its light absorption characteristics
266
to regulate photosynthesis according to light availability in different environments. As a
267
result of this spectacle, the pigment which absorbs the incident wavelengths of light
268
most strongly becomes predominant (Wang et al., 2007). In the present study, the
269
selected strain was capable of complementary chromatic adaptation by synthesizing two
270
different types of phycobilisomes periphery rod depending upon the available light
271
condition. In addition, phycobiliprotein content was significantly correlated with
272
fluorescent white light indicating its important role in cyanobacteria. The present
273
findings are also supported by the work done by Ojit et al. (2015). In the contemporary
274
findings, it was observed that white light was the suitable light source for the enriched
275
production of phycobiliproteins in the selected cyanobacterial strains investigated.
276
According to Ojit et al. (2015) fluorescent white light was the most suitable light source
277
for the enhanced production of phycobiliproteins in almost all cyanobacterial strains. In
278
addition, their study also investigated that when strains were exposed to red light, their
279
PE content decreased but simultaneously phycocyanin (PC) increased when compared
280
to other light qualities. Similarly it was observed in PC rich cultures i.e. in red light, PC
281
quantity decreased and PE production increased in blue light. The chromatic adaptation
282
ability of cyanobacteria comprises of three complex pathway which includes regulation
283
by a phytochrome class photoreceptor (highly responsive to green and red light), a
284
complex two component signal transduction pathway and also reaction based on the
285
redox state (Paliwal et al., 2017). In the present study, higher amount of phycocyanin
286
was found under blue light compared to white light and natural light. Its concentration
287
(0.419 mg mL-1) is also higher compared to the phycocyanin obtained in a study by
288
Ores et al. (2016) on Spirulina sp. LEB 18 (120 µg mL-1). According to Lönneborg et
289
al (1985), phycocyanin content in cyanobacterial cells increase with low light intensity
290
due to the chromatic adaptation ability. Thus, the lower phycocyanin in natural light
291
may be due to the higher light intensity compared to other light sources. Culture under
292
natural condition was exposed to high light intensities (0.02 – 700 µmol m-2 s-1 ) and
293
12:12 h light dark period. According to Raps et al. (1983), with an increase in light
294
intensity, the concentrations of chlorophyll a and phycocyanin decrease. At higher light
295
intensities, the cells lessen the amount of thylakoid membranes and phycobilisomes to
296
decrease the quantity of radiant energy absorbed as possible (Tandeau de Marsac and
297
Houmard, 1993). Another study by Lee et al. (2017) also found lower phycocyanin
298
production when cyanobacterial, Nostoc sp. NK was cultured under white light
299
compared to red and plant fluorescent light. Whereas, similar with the study by Cuellar-
300
Bermudez et al. (2014), phycoerythrin has been found to occur in much lower
301
concentration compared to other pigments because it is mainly affected by changes in
302
pH rather than light.
303 304
Different pH of the medium also affects the phycobiliprotein by changing the
305
culture colour of cyanobacteria. The productions of phycobiliproteins were greatly
306
affected by the pH which might indicate the inability of the cyanobacteria to maintain a
307
constant internal pH. At lower pH 7.5 Anabaena sp. NCCU-9, resulted in reduced
308
phycobiliproteins extraction. This may be due to the extreme buffer’s pH which might
309
cause internal electrostatic attraction by changing the charge on protein giving net
310
positive charge and at this stage protein open up and bound solvent is lost resulting in
311
the denaturation of protein (Roe, 2000).
312
313
Phosphate buffer is expensive, preparation is time consuming and poor purity of
314
the selected cyanobacteria in their final targeted product. Therefore, water is evaluated
315
in this study, as an alternative solvent to extract phycobiliproteins from selected
316
cyanobacteria. In the present study water was used as a solvent to extract
317
phycobiliproteins. It was shown that water solvent had the best extractive performance,
318
not only due to their capacity to extract higher quantity of proteins, but also it
319
circumvents the extraction of chlorophylls. Similarly, Martins et al. (2016) reported
320
that water extract significantly increased the amount of phycobiliproteins when
321
compared
322
conformational structure or chromophore structural integrity.
with
the
conventional
methodology,
without
compromising
their
323 324
The extraction solvent used also affects the allophycocyanin, phycocyanin and
325
phycoerythrin content. The selection of extraction solvent that can lyse the cell wall to
326
increase the seepage of phycobiliprotein pigment is important. In this study,
327
allophycocyanin, one of the pigments production of P. mucicola was significantly
328
higher in all treatments when water was used as the extraction solvent. Whereas, when
329
the cyanobacteria was extracted using phosphate buffer, the most abundant pigment
330
found under phycobiliprotein is the phycocyanin (blue), compared to phycoerythrin and
331
allophycocyanin which is similar to the study by Walter et al. (2011). Hemlata et al.
332
(2011) also reported better phycocyanin and total phycobiliprotein extraction when
333
using phosphate buffer, although with much lower value compared to this study. In
334
terms of total phycobiliprotein production, higher production was obtained in all
335
treatment when P. mucicola was extracted with water, compared to phosphate buffer.
336
This finding is crucial, especially if the extracted phycobiliprotein is intended for human
337
use as it is chemical free.
338 339
In general, the high allophycocyanin, phycocyanin and phycoeryhtrin content
340
and total phycobiliprotein found in P. mucicola (water extract) when cultured in
341
wastewater media might lead to the reduction in the overall cost, especially during mass
342
scale production for commercial purposes. The culture condition, especially nitrogen
343
and carbon sources in the form of culture media, also determines the production of
344
phycobiliprotein by cyanobacteria (Seker and Chandramohan, 2008). In general,
345
cyanobacteria requires nitrogen sources for growth. Cyanobacteria assimilates
346
ammonium ions (NH4+) from external source via an active transport system, while the
347
unprotonated form (NH3) was absorbed by diffusion and is trapped by protonation.
348
Nitrate is taken up by cells via an active transport system before it was reduced to nitrite
349
and then to ammonium (Lioteberg et al., 1996). Under nitrogen depletion, cyanobacteria
350
use phycobiliprotein as a nitrogen source, which can contribute to a decrease in
351
phycobiliprotein production by nblA gene expression (Erikson, 2008). Thus, nitrogen
352
availability plays a very important role in regulating phycobiliprotein and phycocyanin.
353
Eventhough wastewater contain lower amount of nitrogen (TAN and N-NO2), there
354
were no significant decrease in growth and phycobiliprotein content when compared to
355
P. mucicola cultured in BBM. Thus, the wastewater media is capable of providing
356
sufficient amount of nutrient for the cyanobacteria. Collier and Grossman (1994)
357
reported that, deprivation of phosphorus or sulphur can also led to partial or complete
358
decrease of phycobiliprotein by inducing the expression of nblA gene and
359
phycobiliprotein degradation, not only nitrogen.
360
Among the three main pigments of phycobiliprotein, phycocyanin is the most
361
valuable natural blue pigment used in food and pharmaceutical industry due to their
362
colour, fluorescence and antioxidant properties (Kumar et al., 2014). The purity ratio of
363
phycocyanin plays a significant role in commercial applications where it is often
364
evaluated by using absorbance readings. According to Patil et al. (2006), phycocyanin
365
with purity greater than 0.7 is considered as food grade, at ratio 3.9 is considered as
366
reactive grade and above 4.0 as analytical grade. In the present study, higher purity ratio
367
was found in water extract (0.84) of P. mucicola cultured under white light by using
368
wastewater media. The purity ratio was higher than the maximum purity ratio of
369
Spirulina reported by Prabuthas et al. (2011) at only 0.62 purity ratio.
370 371
3.3. Nutrient analysis of wastewater and culture media
372
Wastewater contained sufficient amount of ammonium (6.12±0.01), nitrite (3.80
373
± 0.03), nitrate (3.47 ± 0.02), phosphate (7.20 ± 0.03), calcium (5.40 ± 0.12), potassium
374
(5.45 ± 0.01), sodium (14.25 ± 0.01) and sulphate (5.05 ± 0.02) for culture of
375
microalgae. Physical parameters such as pH (8.25± 0.02), temperature (26.01 ± 0.01)
376
and dissolved oxygen (5.50 ± 0.02) were in the recommended range suitable for
377
cyanobacteria culture.
378 379
Cyanobacteria growth was dependent on a suitable supply of essential
380
macronutrients (nitrogen and phosphorus) but excess nutrients may have toxic effects
381
on the cell metabolism. Excessive nutrients content in commercial media may perhaps
382
affected the selected cyanobacteria growth in the present study. On the other hand,
383
nitrogen and phosphorous are the major nutrients required to grow microalgae. In this
384
study, aquaculture wastewater ensured sufficient amount of nitrogen and phosphorous
385
for proper growth of cyanobacteria. This would facilitate the removal of nutrients from
386
wastewater and production of microalgae biomass concurrently Abdulsada (2014).
387 388
Table 1 showed the nutrient analysis of wastewater before P. mucicola culture and
389
after P. mucicola was harvested. In the present study, both P. mucicola culture by using
390
BBM and wastewater showed a decrease in the nitrite, phosphorus and ammonia
391
content after the culture were harvested. When cultured using wastewater, the highest
392
TAN and N-NO2 uptake of P. mucicola was under white light treatment at 0.142 mg L-1
393
and 1.539 mg L-1, respectively. Initially, the TAN and N-NO2 content of wastewater
394
media was at 6.32 ± 0.01 mg L-1 and 6.50 ± 0.01 mg L-1, respectively. Meanwhile, when
395
P. mucicola was cultured using BBM media, the highest TAN uptake was observed
396
under natural light and N-NO2 uptake was higher under white light at 0.205 mg L-1 and
397
5.424 mg/L, respectively. The initial TAN and N-NO2 of BBM was at 0.483 mg L-1 and
398
12.990 mg L-1. No significant difference (P > 0.05) was found in the P-PO4 content
399
before and after culture of P. mucicola under all treatments.
400 401
Cyanobacteria have developed highly and efficient uptake and retention
402
mechanisms for three nutrient anions: bicarbonate, nitrite, and phosphate (Badger and
403
Price, 2003). P. mucicola culture under white light treatment and natural light treatment
404
utilized most nitrite in both waste water and BBM treatments. However, in blue light
405
treatment, the high nitrite and phosphorus level after harvest in both waste water and
406
BBM may be caused by the slow growth and nutrient uptake by the P. mucicola cells.
407
According to Hu et al. (2000), the nitrate uptake rate increased proportionally with
408
increasing light intensity or parallels to photosynthetic activity. In this study, blue light
409
have lower light intensity compared to white and natural light. Thus, the culture of P.
410
mucicola by using wastewater under white light treatment may serve as an effective
411
way to treat and reuse wastewater.
412 413
Conclusions
414
The growth, biomass yield and phycobiliprotein production as well as purity of
415
P. mucicola varied when cultured under different light sources and media. High growth,
416
chlorophyll a concentration, biomass and phycobiliprotein production were found when
417
P. mucicola was cultivated using wastewater under white light source. Moreover, it was
418
found that extraction of phycobiliprotein by using water, which is chemical free,
419
produced higher phycobiliprotein concentration compared to phosphate buffer. Further
420
studies are warranted for the confirmation and validation of other environmental factors
421
suitable for the production of phycobiliproteins in cyanobacteria.
422 423
Acknowledgement
424 425
This study was supported by the Ministry of Higher Education, Malaysia, through
426
Fundamental
Research
Grant
427
FRGS/1/2015/WAB01/UMT/03/6.
Scheme
(FRGS)
project
No.
428 429
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platensis under different light spectra. Braz. Arch. Biol. Techn. 54, 675-682.
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569
Figure captions
570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602
Figure 1. Optical density (abs) versus culture period (day) for P. mucicola cultured under different media (wastewater (WW) and BBM) and light sources (white light (WL), blue light (BL), natural light (NL)). Values are mean ± standard error. Figure 2. Phycobiliprotein pigment production (mg mL-1) of P. mucicola cultured under different media, light sources and extraction solvents.. Values are mean ± standard error. Figure 3. Total phycobiliprotein production (mg mL-1) of P. mucicola cultured under different media, light sources and extraction solvents. Values are mean ± standard error. Figure 4. Purity of phycocyanin from cyanobacteria cultured under different media, light sources and extraction solvents. Values are mean ± standard error.
Optical density (abs)
0.8
BBM WL
0.7
BBM BL
0.6
BBM NL WW WL
0.5
WW BL 0.4
WW NL
0.3 0.2 0.1 0 0
603 604 605
Fig. 1.
1
2
3 4 Culture period (Day)
5
6
7
606 607 0.600
Pigment production (mg mL-1)
BBM WL 0.500
BBM BL BBM NL
0.400
WW WL WW BL
0.300
WW NL 0.200 0.100 0.000 Phosphate buffer
Phycocyanin (PC)
608 609 610 611 612 613 614 615 616 617 618 619 620
Distilled water
Fig. 2.
Phosphate buffer
Distilled water
Allophycocyanin (APC)
Phosphate buffer
Distilled water
Phycoerythrin (PE)
621 622 623 624 625
Total phycobiliprotein (mg G-1)
626 Phosphate buffer
300 250 200 150
100 50 0
WL
BL BBM
627 628 629 630 631 632 633 634 635 636 637 638
Fig. 3.
Water
NL
WL
BL WW
NL
639 640 641 642 643 644 645 646 647 648 1.4
Phosphate buffer
Water
1.2
Purity
1.0 0.8 0.6 0.4 0.2 0.0 WL
BBM
649 650 651 652
BL
Fig. 4.
NL
WL
BL WW
NL
653
Table captions
654
Table 1
655
Nutrient analysis of wastewater before P. mucicola culture and after P. mucicola
656
was harvested. Values are means ± standard errors (n=3).
Culture
Nitrite nitrogen
Phosphate
nitrogen (TAN)
(NO2-N)
phosphorous (P-PO4)
(mg L-1)
(mg L-1)
(mg L-1)
Light
Sample media
Total ammonium
source
Before
Wastewater
-
6.32 ± 0.01
6.50 ± 0.01
5.53 ± 0.06
culture
BMM
-
0.48 ± 0.02
13.09 ± 0.01
11.06 ± 0.03
WL
0.04 ± 0.01
1.53 ± 0.04
4.00 ± 0.01
BL
0.06 ± 0.03
3.25 ± 0.01
4.40 ± 0.01
NL
0.07 ± 0.01
1.32 ± 0.03
5.00 ± 0.08
WL
0.44 ± 0.01
5.42 ± 0.01
10.50 ± 0.01
BL
0.38 ± 0.00
11.64 ± 0.02
10.01 ± 0.02
NL
0.20 ± 0.03
8.10 ± 0.01
9. 80 ± 0.05
Wastewater After harvest
BBM
657 658 659
660 661
662 663
Optical density of cyanobacteria under different light sources and media
Chlorophyll a of cyanobacteria under different light sources and media
Biomass of cyanobacteria under different light sources and media
664 665
Biomass of cyanobacteria was harvest at stationary phase & freeze dried
666 667 668 669
Dried biomass of cyanobacteria was extracted with water and phosphate buffer for phycobiliprotein production
670 671
672 Phycobiliprotein pigment production of cyanobacteria cultured under different media, light sources and extraction solvents
Total phycobiliprotein production of cyanobacteria cultured under different media, light sources and extraction solvents
Purity of phycocyanin from cyanobacteria cultured under different media, light sources and extraction solvents solvents
673
Research highlights
674 675 676 677 678 679 680 681 682 683 684 685 686 687 688
Effect of different light sources and media on the growth of cyanobacteria was determind Effect of different light sources and media on the phycobiliprotein production of cyanobacteria Pseudanabaena mucicola grown in wastewater under white light had significantly higher biomass Cyanobacteria biomass extracted with water had higher production and purity of phycocyanin. Phycobiliprotein extracted using water from P. mucicola can be food grade natural blue pigment. Cyanobacteria grown in wastewater could cut down the production cost of phycobiliprotein.