OPTIMIZATION OF CYANOBACTERIA B GROWTH BASED ON

NITROGEN AND VITAMIN B12 CONTENT

WAN NURUL EZZATI FARHANI BINTI ISHAK

Written Proposal Submitted in

Partial Fulfilment of the Requirements for the
Degree of Bachelor of Science (Hons.) Biology
in the Faculty of Applied Sciences
University Teknologi MARA

DECEMBER 2019
 TABLE OF CONTENTS

PAGE

CHAPTER 1 INTRODUCTION
1.1 Background Study 2
1.2 Problem Statement 4
1.3 Significance of the Study 5
1.4 Objectives of the Study 6

CHAPTER 2 LITERATURE REVIEW

2.1 Cyanobacteria 7
2.2 Cyanobacteria in Bioremediation 9
2.3 Augmentation of Cyanobacteria Growth based on
Nutrient Alteration
2.3.1 Optimization of vitamin B12 concentration in 11
cyanobacterial growth
2.3.2 Optimization of nitrogen and phosphorus content 13

CHAPTER 3 METHODOLOGY
3.1 Materials
3.1.1 Chemicals 16
3.1.2 Equipment and Apparatus 17
3.2 Methods 17
3.2.1 Preparation of BG-11 medium 18
3.2.2 Growth Curve Study of Cyanobacteria 18
3.2.3 Preparation of Cyanobacteria Culture 20
3.2.4 Effect of different Nitrogen Concentration on 21
Cyanobacteria Growth
3.2.5 Effect of different Vitamin B12 Concentration 22
on Cyanobacteria Growth
3.2.6 Measuring Cyanobacteria Growth using the Pour 22
Plate Method
3.3 Statistical Analysis 25

CITED REFERENCES
GANTT CHART
STUDENT-SUPERVISOR MEETING FORM
1
 CHAPTER 1

INTRODUCTION

1.1 Background of Study

Cyanobacteria which is also known as the blue green algae is a type of

prokaryotes that are able to synthesis their own food through

photosynthesis. They are commonly known as blue green algae as they

possess chlorophyll and algal-like appearance but further studies have

classified them as prokaryotes. According to Singh et al. (2019)

cyanobacteria are distinctive photosynthetic prokaryotes that showed

versatile nature to sudden change and diversification towards light,

temperature, salinity and nutrient composition. In addition, cyanobacteria

are present in widespread area of habitat including marine, soil,

freshwater, volcanic ash, salted soils, disturbed area and can be found in

terrestrial system.

Bioremediation is a technique that used microorganism such as bacteria to

degrade contaminant in the environment and hazardous waste. According

to Coelho et al. (2015) bioremediation, with the use of microorganisms,

2
has tremendous potential for future development due to its environmental

impact and potential cost-effectiveness. The reason why microorganism

bioremediation is much favoured by scientists as opposed to conventional

method is due to the rapid action of microorganisms on contaminants even

when they are present in very diluted solutions and also because it is green

and socially tolerable by the environment. Moreover, cyanobacteria are

able to adapt to extreme conditions thus it works more efficiently to treat

stubborn heat-resistant pollutants. Globally, cyanobacteria have been

recognized for their potential uses in food, fertilizer, fuel ,vitamins,

enzymes pharmaceuticals toxin, pollution abatement and many more

(Karn, 2016).

Nitrogen availability is needed for cyanobacterial growth as noted by Das

and Sharma (2015). Despite this, it was found out that nitrogen starvation

is often studied as an effective method to induce the accumulation of lipids

in micro algal cells. Nonetheless, Zhai et al. (2017) stated that medium

components in wastewater have been shown to have a major impact on

microalgal production, biomass components and nutrient uptake, whereas

low nutrient content generally results in low productivity of biomass,

limiting the integration of microalgal cultivation with wastewater

treatment.

3
 However, some cyanobacteria species still are not able to grow well even

though they have been supplied with basic necessities such as inorganic

nutrients. These occur as nearly half of marine and freshwater species,

especially cyanobacteria, require exogenous B vitamins such as vitamin

B1 (thiamine), B7 (biotin) and B12 (cobalamin) to grow apart from the

inorganic nutrients supplied such as phosphorus and nitrogen.. Studies

done by Edelmann et al. (2019) concluded that Spirulina sp. are unable to

grow in the absence of B vitamins even though light and inorganic

nutrients are present. Subsequently, Bohutskyi et al. (2019) stated that

vitamin B12 could be used for a more sturdy algal bioprocesses design that

will retained high biomass productivities especially under stress

conditions.

1.2 Problem Statement

Upon many studies surrounding cyanobacteria, there have been fewer

studies on the right nutrient modification for optimization of cyanobacteria

growth as many past researches are only focusing on the general outline

for the optimal cyanobacteria growth parameters without coming out with

a well-devised and structurally accurate composition of cyanobacteria

4
 growth parameters needed. Likewise, although there are already various

studies done on cyanobacteria yet the accurate essential growth

modification requirement needed for each different bacterial strains to

grow are still undiscovered as different species in cyanobacteria have

different requirement and tolerance. Hence, identifying what are the most

accurate growth requirement needed to facilitate the growth of

cyanobacteria is crucial.

1.3 Significance of the Study

This project will be beneficial because it allows researchers to know the

optimal nutrient optimizations needed to enrich and spread cyanobacterial

isolates and how it can help improve the natural biodegradation behavior

of the purified isolates. Moreover, it can act as a reference for future

studies on the effect of modified nutrient modifications in cyanobacteria

for other group of microorganisms. Furthermore, the identification of the

perfect nutrient parameters leading to the augmentation of mass

cyanobacteria productions could help future researcher in optimization of

cyanobacterial growth. Other than that, the identification of the right

nutrient modifications could also be a stepping-stone for enhancing mass

production of cyanobacteria to generate high-value products such as

biofuels, natural cyanobacterial products (e.g. sugars and isoprene), and

other commodity chemical. Thus, detailed research into the properties of

5
 cyanobacteria that can help in bioremediations can be intensified. This

study are expected to help others in finding out what is the most suitable

conditions needed for cultivating mass growth of cyanobacteria so that it

can easily be extracted to remediate polluted environments.

1.3 Objectives of the Study

The objectives of the study are:

1. To assess the effect of different nitrogen level on the growth rate

of cyanobacteria B

2. To access the effect of doubling vitamin-B12 in the growth of

cyanobacteria B.

6
 CHAPTER 2

LITERATURE REVIEW

2.1 Cyanobacteria

Cyanobacteria, also known as blue green algae, are a large and

morphologically diverse group of prokaryotic microorganisms.

Cyanobacteria contribute approximately 15% of the total algal flora in the

tropical regions and about 2% in the temperate regions. According to Rishi

and Awasthi (2015), cyanobacteria are found as photoautotrophs,

photoheterotrophs, chemoheterotrophs and bacteria like anoxygenic

photosynthesis at different condition. Some cyanobacteria have the ability

to fix atmospheric nitrogen through special cells called heterocysts

(Gautam et al., 2019)

Other than that, some cyanobacteria have dexterity to survive in aberrant

habitats like low and high pH, high temperature, metal polluted soils and

waters and in such environment where other algae could not be survive

(Rishi and Awasthi, 2015). Cyanobacteria are omnipresent as they are an

enormously diverse group of photosynthetic prokaryotic microorganisms

7
whose have adaptive capacity along with the ability to tolerate extreme

conditions (Gaysina et al., 2019; Prihantini et al., 2019) Cyanobacteria can

be found in extreme environments because they can withstand high

temperatures. Cyanobacteria that can acclimatize at high temperatures

have metabolic processes associated with thermostability (Prihantini et al.,

2019).

In addition, Mur et al. (1999) claims that the capability of cyanobacteria

to colonize infertile substrates such as volcanic ash, desert sand and rocks

is remarkable. Cyanobacteria are often the first microorganisms to

colonize that kind of barren habitats. Besides, many cyanobacteria species

are capable of living in soils and other terrestrial habitats, where they are

key to the functioning of ecosystems and the cycling of nutrients as noted

by Mur et al. (1999). Cyanobacteria had ultraviolet absorbing sheath

pigments that would increase their fitness in a relatively exposed soil

environment. Likewise, cyanobacteria have the capability to outlast in

different types of environments due to their ability to change their mode of

nutrition (Rishi and Awasthi, 2015).

Furthermore, Mur et al. (1999) showed that limnic and marine

environments are a prominent biosphere of cyanobacteria. Cyanobacteria

8
 thrive in salty, brackish or fresh water, in cold and hot springs and in

environments where no other microalgae can survive. Most marine forms

thrive along the coast as benthic vegetation in the zone between high and

low tide marks.

According to Patel et al. (2017), there are several benefits on the use of

cyanobacteria as biofuel agents such as the minimum to trivial use of the

arable land and positive role in reducing the elevated atmospheric CO2

levels. Therefore, rational utilization of the cyanobacteria could contribute

in gathering the dire needs of energy. Above all, cyanobacteria are good

agents to provide environmental refunds for pollution.

2.2 Cyanobacteria in Bioremediation

Bioremediation can be defined as any process that uses microorganisms,

fungi, green plants or their enzymes to return the pollutant-affected natural

environment to its original productive and fertile form. Bioremediation

using oxygen-evolving cyanobacteria is a cost-effective and eco-friendly

approach to environment management. Moreover, bioremediation process

aims towards the eradication of toxic wastes effluents released from

industries or accumulated to the fields along with chemical fertilizers.

Thus, cyanobacteria had been proposed as the integral bio-agents in

9
ecological reclamation of deteriorated lands (Singh et al., 2016).

Moreover, cyanobacteria could act as bioremediators because they had

some superiority over other microorganisms due to their photoautotrophic

nature and capacity to fix atmospheric nitrogen, which makes them self-

sufficient to survive in polluted and highly polluted environments for

growth, sustenance and ambibidexterity. (Sokhoh et al., 1992).

Adding to that statement, Quintana et al. (2011) stated that cyanobacterial

bioremediation is based on the notion to accomodate complex pollutants

from cyanobacteria surroundings and use them to enhance for their

amplification and metabolism. It could also help to restore cyanobacteria

species from a lethal to non-lethal form. An effective tool for sustainable

development and pollution control is the cyanobacterial strategy for the

eradication of pernicious pollutants. Cyanobacteria can overcome

secondary contamination because they can remedy radioactive materials,

petroleum waste and eradicate pesticide toxins as noted by Singh et al.

(2019).

Likewise, Singh et al. (2019) also found out that cyanobacteria are useful

for wastewater treatment. These discoveries occur due to cyanobacteria

capability to destroy numerous toxic compounds, including pesticides.

10
 Besides, the processes of bioaccumulation and bio-absorption can

removed metals from effluents. Above all, bioremediation of algal is an

environmentally friendly, cost-effective and accurate method for

addressing environmental problems.

2.3 Augmentation of Cyanobacteria Growth based on Nutrient Alteration

2.3.1 Optimization of vitamin B12 concentration cyanobacterial growth

Microalgae are a diverse group of unicellular organisms comprising of

eukaryotic protists, prokaryotic cyanobacteria, and blue-green algae (Day

et al., 1999). Some prokaryotes, including microalgae, require exogenous

B vitamins for development, such as vitamin B1 (thiamine), B7 (biotin),

and B12. Grossman (2016) has shown that exogenous vitamin B12 is

required as a cofactor for different enzymatic reactions for marine and

freshwater microalgae, like many other prokaryotes.

Studies done by Grossman (2016) demonstrate that microalgae acquire

vitamin B12 from marine prokaryote. Vitamin B12 is synthesized as

pseudocobalamin by planktonic cyanobacteria, a non-bioactive element of

microalgae. However, many microalgae can restructure pseudocobalamin

to the active form of cobalamin, adding complexity to the environmental

11
assessment of active vitamin B12. Thus, the availability of thiamine, biotin

and cobalamin as noted by Croft et al. (2005) may restrict the growth of

microalgae. Hence, Croft et al. (2005) claims that microalgae that do not

manufactured vitamin B12 de novo but still require it for metabolism may

acquire it via a mutually beneficial relationship with cyanobacteria. In

addition, Matthews (2009) said that vitamin B12 is a crucial micronutrient

co-factor for different biosynthetic functions in nearly all living cells, even

though cobalamin is only synthesized by several species of cyanobacteria..

Besides, Vitamin B12 also plays a vital role in pigment and membrane

biogenesis in photosynthetic microorganisms (Croft et al., 2005).

Furthermore, Bohutskyi et al. (2019) claims that static Cyanobacterium

stanieri cultures grown in the absence and presence of cobalamin formed a

loose, benthic biofilm over a 42-day duration. Thus, cobalamin

supplementation has affected the morphology of the formed biofilm and

the amount of accumulated biomass event, although Cyanobacterium

stanieri does not need cobalamin to grow. Subsequently, the establishment

of complex microbial systems is recognized as a key factor in cobalamin

cross feeding as noted by Bohutskyi et al. (2019). Alternatively, the results

of Trichodesmium s.p growth observed by Rodriguez and Ho (2015) stated

that cobalamin was independent of B12 concentration supply in media

12
 with sufficient cobalt; however, when Co was insufficient, cobalamin was

still crucial for diazotrophic growth.

These findings show that for Trichodesmium sp. optimum growth, vitamin

B12 may be synthesized in cultures or the associated bacteria may be used

to produce B12. Hence, the trends observed in the validation experiments

show that the availability of Vitamin B12 affects Trichodesmium sp's

nitrogen-fixing capacity (Rodriguez and Ho, 2015). Accordingly,

Bohutskyi et al. (2019) reported that further understanding and mastery of

natural oxidative stress resistance cobalamin-modulated mechanisms could

be used to design and implement more robust algal bioprocesses that

maintain high biomass productivity, particularly under stress conditions.

2.3.2 Optimization of nitrogen and phosphorus content

Nitrogen (N) and phosphorus (P) are natural elements that are essential for

flora and fauna to grow, mature and reproduce. Consequently, Parrish

(2014) stated that many previous studies have shown that optimizing the

rate of nutrient intake such as nitrogen and phosphorus would affect the

growth rate of cyanobacteria and may result in increased yields of biomass

or lipids.

13
Cyanobacteria such as Microcystis aeruginosa sp dominate when N

concentrations are sufficiently low to be considered the nutrient limiting

factor. Subsequently, absolute and relative nitrogen and phosphate

concentrations influence the growth rate, abundance and composition of

phytoplankton in lakewater as noted by Giannuzzi (2019). The levels of

nitrogen and phosphate in terms of its trophic condition at the time of

measurement is commonly measured as the total biomass weight in the

water body . Generally, the lake's trophic state rises with increases in term

of the total nitrogen (TN) and total phosphorus (TP) concentrations.

According to Giannuzzi (2019), the elevated phosphorus content in

Korea's Nakdong River is believed to be the main cause of Microcystis

aeruginosa sp. Besides, Zhai et al. (2017) states that additionally a

phosphate concentration of 250 mg / L in the form of K 2HPO4 is needed to

maximize the production of biomass. Moreover, report showed that the

existence of nitrogen-fixing cyanobacterium in water bodies makes it

unlikely that nitrogen will be the limiting factor of blooms. Nevertheless,

Parrish (2014) found that nitrogen has a much greater influence on the

growth and functions of Microcystis aeruginosa sp. compared to

phosphorus. However, Microcystis aeruginosa still uses phosphorus to

sustain itself, although the roles of phosphorus in the growth, toxicity, and

distribution of Microcystis aeruginosa are not all similar.

14
In addition, De Marsac and Houmard (1993) showed that in order to deal

with changes in ecological conditions, cyanobacteria have established

unique physiological and morphological characteristics such as nutrients

and light. According to Yenkie et al. (2016), the nitrate absorption rate

rose proportionally with rising light magnitude or correspondent to

photosynthetic event. The variation in the concentration of nitrogen in

cultivation medium that is related to light intensity influences the

elemental and biochemical content of microalgae biomass (Yenkie et al.,

2016).

In addition, for development, metabolism and production of chlorophyll

and lipids in algal cells, the configuration and quality of carbon and

nitrogen sources throughout the medium is important . Das and Sharma

(2015) has found out that nitrogen deprivation is often treated as an

effective approach to induce lipid aggregation in microalgal cells. Lipid

accumulation in algal cells was 78% higher than before optimization as

noted by Ye et al. (2018). Furthermore, the use of the optimization

methods for cyanobacteria cultivation under nitrogen-limited parameters

resulted in elevated biomass and lipids output in pilot cultivation in a flat

panel photobioreactor. Similarly, Yenkie et al. (2016) also claims that

15
nitrogen limitation would limit biomass titer thus substantially increasing

downstream processing costs.

16
 CHAPTER 3

METHODOLOGY

3.1 Materials

3.1.1 Chemicals

The medium that will be used are BG-11 modified medium (SP). This

medium consisted of solutions A and B containing (in grams): A; sodium

bicarbonate (NaHCO3) 11.61 g, sodium carbonate (Na2CO3) 3.53 g,

potassium phosphate (K2HPO4) 0.5 g dissolved in 500 ml distilled water

whereas B; sodium nitrate (NaNO3) 5 g, potassium sulphate (K2SO4 )1 g,

sodium chloride (NaCl) 1 g, magnesium sulphate heptahydrate

(MgSO4.7H2O) 0.2 g, calcium chloride (CaCl2) 0.04 g, and 1 ml

Ethylenediaminetetraacetic acid (EDTA) (0.5 M). Other than that,

micronutrient solution (CHU no.10) is also needed. Micronutrient solution

consisted of the following trace metals (in milligrams) dissolved in one

litre distilled water: disodium salt dehydrates (Na2-EDTA) 50 g, boric acid

(H3BO3) 618 g, copper sulphur pentahydrate (CuSO4.5H2O) 19.6 g, zinc

sulphate heptaydrate (ZnSO4.7H2O) 44 g, calcium chloride hexahydrate

(CaCl2.6H2O) 20 g, Manganese(II) chloride (MnCl2) 12 g and molybdic

acidsodium salt dehydrate (Na2MoO4 · 2H2O) 12.6 g.

17
3.1.2 Equipment and Apparatus

The lists of equipment that will be used are the spectrophotometer, laminar

air flow chamber, 0.22-mm polycarbonate membrane, refrigerator,

incubator, micropipettes and autoclave machine. Lists of apparatus that

will be used are gloves (medium size), blotting paper towel, micropipette

of: 200µl-1000µl, 20µl-200µl, 2µl-20µl, 500 ml Schott bottle, Petri dishes,

conical flasks of 250ml and 500 ml, beaker, spatula inoculating loop,

measuring cylinder of 10 ml and 50 ml, and parafilm roll.

3.2 Methods

As shown in the flowchart, procedures that will be used for this study are

the preparation of BG-11 medium, growth curve of cyanobacteria,

preparation of cyanobacteria culture, determine the effect of nitrogen

concentration on cyanobacteria growth, determine the effect of vitamin B-

12 concentration on cyanobacteria growth and measuring cyanobacteria

growth using pour plate method.

18
3.2.1 Preparation of BG-11 medium

To produce BG-11 modified medium, solutions A and B will be sterilized

by autoclaving separately at 121oC for 20 min. Micronutrient solution will

be sterilized by filtration through a 0.22-mm polycarbonate membrane to

avoid interaction and precipitation of heavy metals. After sterilization,

solutions A and B will be combined and 1 ml of micronutrient solution

and 1 ml of vitamin B12 (stock solution 15.0 x 10-6 g) will be added.

3.2.2 Growth Curve Study of Cyanobacteria

Cyanobacteria B will be sub-cultured from the existing cyanobacteria agar

in the laboratory into BG11 medium liquid. Next, the samples will be

grown by adjusting the light:dark cycle, with 16:8 hours of white light 8

feet 40 W with light intensity 3200 lux, the temperature at 25 to 30 oC, and

agitation at 100 rpm for 7 days. All of these adjustments done will lead to

the enhancement of mass production and it will be used to determine the

optimum time for harvesting cyanobacteria.

Cyanobacteria culture growth rate will be observed using turbidimetric

determination via optical density (OD) measurement using a

spectrophotometer. Turbidimetric determination is useful as it uses a

spectrophotometer to track changes in optical density (OD) over time to

19
plot the growth curves of cyanobacteria in broth or liquid media. Turbidity

measurements rely on the fact that the higher the cell concentration, the

higher the turbidity. In other words, the transmission of photons via the

sample would decrease as the cell count in a sample increases.

Growth curves were measured by determining the optical density at 750

nm (OD750). The standard cycles of bacterial culture development (lag,

log, stationary, and death) will be recorded, with the mid-log stage being

known as the period where cyanobacteria divide as quickly as possible.

Bacterial OD750 applications are used to determine the optimal time at

which the competent cells that would be extracted will be harvested and

prepared.

Turbidimetric determination will be used to construct a cyanobacteria

growth curve. It utilizes spectrophotometric measurements twice a day for

a week. Firstly, sterile broth or liquid media that will be used for

inoculation will be prepared. The spectrophotometer will be turned on and

allowed to warm up, preferably for several minutes before it can be its use.

The the spectrophotometer wavelength will be set to 750 nm. Then, 5 mL

of uninoculated sterile media or sterile distilled water depending on the

circumstances will be added to a clean cuvette and it will act as blank. The

20
 blank will be put in a spectrometer and will be set to 0 ABS. This step

streamlines the turbidity of the empty media so that further measurements

can accurately measure cyanobacteria development causes by changes in

the turbidity. Next, the primary substrate from prepared liquid media will

be inoculated. Immediately after inoculation, a 5 mL specimen of the

inoculated medium will be obtained and piped into a sterile cuvette. It

would then be positioned in the blank spectrophotometer, and the OD

measurement will be documented at "0" time. The readings will be

recorded and tabulated. Lastly, the readings will then be plotted on a graph

with Time as the X-axis and OD as the Y-axis. Turbidimetric

determination is very useful when drawing a generic growth curve; as it

helps one to accurately chart shifts in growth phases without the

complexity of counting plated colonies.

3.2.3 Preparation of Cyanobacteria Culture

Cyanobacteria B will be sub-cultured from the existing cyanobacteria agar

in the laboratory into BG11 medium liquid. The optimum time for

cyanobacteria B to be sub-cultured is based on the growth curve study

done above. The samples will be grown by adjusting the light:dark cycle,

with 16:8 hours of white light 8 feet 40 W with light intensity 3200 lux,

the temperature at 25 to 30oC, and agitation at 120 rpm. All of the

adjustments done will lead to the enhancement of mass production. After

21
 that, the cyanobacteria culture will be poured into the agar medium and

left to solidify. Then, the colony forming unit (cfu) will be calculated by

using pour plate method. Cyanobacteria culture harvested will then be

used to determine the effect of different nutrient modifications on

cyanobacterial growth.

3.2.4 Effect of Different Nitrogen Concentration on Cyanobacteria Growth

To attain optimal growth of these cyanobacteria, the evaluation of suitable

media is felt to be the prime requisite. One of the factors that can affect the

cyanobacterial growth is nitrogen content. BG-11 medium will be

modified by intensifying the nitrogen concentration as the enzymes

accountable for dechlorination activities (nitrate reductase) are prompt at

high nitrogen levels (Kuritz et al., 1997). The control medium for nitrogen

concentration will consist of 1mL diluted cyanobacteria cultured in 10mL

cyanobacteria growth medium. The concentration of nitrogen content used

will be varied from 0.03, 0.10 and 1.50 (mM). Each of the concentration

will be in triplicate of 1 ml diluted cyanobacteria cultured medium and 10

ml of modified BG-11 medium.

Cyanobacteria cultures will then be harvested in triplicate for each

treatment at five pre-determined time points (day 0, 3, 6, 9 and 12). During

22
 the harvesting process, 1 mL of each cyanobacteria triplicate cultures will

be diluted in 9 mL distilled water. Then, the diluted cyanobacteria colonies

will be observed using the pour plate method where it will be used to

count their colony forming units per millimetre (CFU/mL).

3.2.5 Effect of Different Vitamin B12 Concentration on Cyanobacteria

Growth

Another modification for BG-11 medium will be by using different levels

of vitamin B12 concentration, which hypothesized to enhance the

cyanobacteria growth. The control medium for vitamin B-12 concentration

will consist of 1mL diluted cyanobacteria culture in 10 mL modified BG-

11 cultured media of cyanobacteria with fix nitrogen content (10mM) but

the modified BG-11 cultured media growth for nitrogen modification will

exclude the 1ml Vitamin B-12. The amount of Vitamin B12 that will be

applied to the cyanobacteria culture will be in three different

concentrations (0, 10, 40,100 pmol L-1). During the harvesting process, 1

mL of each triplicate cyanobacteria cultures will be diluted in 9 mL

distilled water. Then, the diluted cyanobacteria colonies will be observed

using the pour plate method where it will be used to count their colony

forming units per millimetre (CFU/mL).

3.2.6 Measuring Cyanobacteria Growth using Pour Plate Method

23
Before measuring cyanobacteria growth using pour plate method, serial

dilution of cyanobacteria culture will be done. Serial dilution is a series of

sequential dilutions used to reduce a dense culture of cells to a more usable

concentration. Each dilution will reduce the concentration of bacteria by a

specific amount. Count range of bacteria between 30 to 300 bacteria

colonies per plate are preferred. This to ensure a countable plate can be

obtained to calculate colonies form unit per ml (CFU/mL). A 10 fold of

dilution is commonly practise and will be used in this experiment.

The following steps will be used to conduct serial dilution;1)a set amount

of the initial 1.0 mL triplicate cyanobacteria culture will be applied to and

thoroughly blended with the first dilution pipe solution comprising of 9.0

mL sterile distilled water. This process reflects a dilution ratio of 10 or

1:10 compared to the previous culture; 2) The same amount, 1.0 mL, will

be extracted from the very first dilution and combined with a fresh pipe of

9.0 mL of dilution solution. Compared to the previous culture the dilution

level is now 1:100; 3) This cycle will continue until a series of dilutions

are produced to support the required concentration needed of cells for

accurate measuring. Each step results in a further 10-fold change in the

concentration from the previous concentration. By calculating the total

dilution over the entire series, it is possible to know how many bacteria

have at the beginning.

24
After that, the number of cells in culture will be quantified by the pour

plate technique, which is typically the preferred route for counting the

number of colony-forming bacteria in a fluid sample. In pour plate

method, a set amount of inoculum usually 1 ml from the diluted samples

will be mounted in the center of the sterile Petri dish using a sterile pipet.

Approximately 15mL of molten cooled agar will then be poured into

inoculum-containing growth medium and mixed well. The plate will be

inverted and incubated at 37 °C for 24-48 hours after the agar solidified.

The colonies of cyanobacteria will appear on the substrate and in the

media. Supposedly, each colony that will be formed represents a "colony-

forming unit" (CFU). Colony-forming units of the cyanobacteria will be

counted and their abundance will be calculated as CFU/mL. CFU/mL will

be used to show the percent distribution for the bacteria colony formed in

each different location.

The CFU/ml can be calculated using the formula:

The number of CFUs per ml of sample =

The number of colonies (30-300 plate) X The dilution factor of the plate

counted.

25
3.3 Statistical Analysis

Statistical analyses that will be used is one-way ANOVA alongside a post

hoc analysis [Tukey's honestly significant difference (HSD). For one way

ANOVA, the data on cyanobacteria B growth will be expressed as mean ±

S.D. The mean values and standard deviation will be calculated based on

the triplicate values. The effects of growth factor, which are vitamin B-12

and nitrogen at three different concentrations on cyanobacteria growth

rate, will be analyzed through ANOVA using SPSS software version 25.

The level of significance was set at 0.05 for all testing conditions.

Meanwhile, the post hoc analysis or also known as Tukey's honestly

significant difference test (Tukey's HSD) will used to test differences

among sample means for significance. It will be used, as ANOVA does

not provide any deeper insights into patterns or comparisons between

specific groups. Thus, the Tukey’s test compares the means of all

treatments to the mean of every other treatment and is considered the best

available method in cases when confidence intervals are desired or if

sample sizes are unequal.

26
 Preparation of BG-11 medium

Growth Curve of Cyanobacteria

Preparation of Cyanobacteria Culture

Effect of Different Nitrogen Effect of Different Vitamin B12

Concentration on Cyanobacteria Growth Concentration on Cyanobacteria Growth

Measuring Cyanobacteria Growth using

Pour Plate Method

ANOVA Test

Figure 3.1 Flowchart of the study

27
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Form 3: FYP Gantt-chart

Sem 1 2
No. Activity Month 1 2 3 4 5 6 7 8
Week 2 4 6 8 10 12 14 2 4 6 8 10 12 14
1. Identify a proposed title x
2. Collection of research materials x x x
3. Literature review x x
4. Identifying methodology x x x
5. Research design planning x x
6. Flowchart of study x x
7. Finalise proposal x
8. Submission of proposal x x
9. Assembly of apparatus x x
10. Conducting experiment x x x
11. Collecting data x x x
12. Analysis of data x x x
13. Presenting results x
14. Final draft of report x x
15. Submission of report x

31
Form 2: Student-Supervisor Journal
FSG/PTA/08/2007/02

Course code :
Document type :
Semester :
Part :
Student name :
UiTM no :
Project title :
Supervisor :
Co-supervisor :

Complete/ Not Reason (If Not Signature

Date Task Due date Action to be taken
Complete Complete) Student Supervisor

32
33