BIOLOGICAL SCIENCE (Plant Biology)

Discovery of a diatom that oozes oil

Vandana Vinayaka,b1, Vichar Mishrab, Shristy Gautamb, Anshuman Raia

and Richard Gordonc

a Diatom Research Unit, Forensic Science Laboratory (Haryana), Madhuban, India, 132037; E-Mails:

kapilvinayak@gmail.com and anshuman.rai@gmail.com

b Department of Criminology and Forensic Science, School of Applied Sciences, Dr. H.S. Gour

University (Central University), Sagar, Madhya Pradesh, India; E-Mail: kapilvinayak@gmail.com,

vicharmishra@gmail.com and shristy.g@gmail.com

c Embryogenesis Center, Gulf Specimen Marine Laboratory, 222 Clark Drive, Panacea FL 32346

USA; E-Mail: DickGordonCan@gmail.com

Abstract: Diadesmis confervacea produces substantial amounts of oil which could be utilized as

biofuel. We discovered that oil exocytoses spontaneously from cells of D. confervacea, a new

observation for diatoms. Morphological observation shows about 50% of cultured diatoms oozing oil

by the 10th day of culture and about 80% oozing by the end of a month. When the 31st day plates were

subcultured on fresh media they resumed active growth, division and oil production and oozing in a

similar manner over the following month. GCMS (gas chromatography–mass spectrometry) results

showed formation of free fatty acids at retention time peak 12.91 minutes and FAME (fatty acid

methyl esters) at retention time peak 18.32 minutes upon transesterification. Spontaneous oozing

greatly reduces the cost of algal fuels, as separation of the oil from the cells has hitherto been an

expensive, fuel requiring process, and makes it easier to design diatom biofuel solar panels.

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Keywords: diatom oil, lipids, diatom solar panels

Significant statement: Since diatoms have abundant oil but to extract oil from them is tedious and

cost effective process. Though the idea of increasing oil accumulation in diatoms by genetic

engineering is quite successful and achieved but if nature already has in its stock certain oil rich

diatoms which when cultured are loaded fully with oil acquiring the shape of whole cell, the need of

genetic engineering and its cost can be reduced. We have discovered a strain of diatom Diadesmis

confervacea which not only accumulates high amount of oil (14.6%) but also extracts oil naturally near

31st day of culturing in vivo when the cells attains maturity which is very significant in the area of

economical biofuel production.

\body

Footnote:

VV conducted, monitored and wrote the paper, SG collected the water sample, VM and SG did the

experimentation, AR analyzed the technical data. RG guided the whole experimentation, edited and

checked the ms.

1 Author to whom correspondence should be addressed; E-Mail: kapilvinayak@gmail.com;

Tel.: +91-91-79-577953

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1. Introduction

The global economy suffers due to the price hike in fuels time to time, which are obtained from natural

reservoirs. Diatom oil production is expected to be 7 to 200x per hectare compared to oilseed crops [1,

2, 3]. Diatoms play an important role in the ecological balance of earth because of their carbon dioxide

fixing ability and are responsible for “~20-25% of the world net primary production” [4] (cf.: [5, 6]).

When the silica shell is discounted, the dry weight of oil per dry weight of cell may exceed that of any

other organism [7]. Geologists suggest that fossil diatoms could be the source of much of the world’s

crude oil [8]. Mass culturing of diatoms for biofuel would not compete with food production [9] nor

contribute to net increase in atmospheric CO2. Diatoms include species able to survive in diverse

conditions [10, 11, 12] including high temperature [13, 14, 15, 16, 17].

It has been reported that when diatoms are put under stress conditions (low nutrients, high temperature,

low silica, high or low light, etc.) the concentration of oil increases [18]. This may be due to the shift

in lipid metabolism from synthesis of intracellular plasma membrane to storage lipid. Genetic

transformation of diatoms [19, 20, 21, 22, 23, 24, 25, 26, 27] or selective breeding [28] might someday

further increase oil production under broader conditions. In this paper we have sought diatoms with

high oil content in vivo under natural conditions and found one species that exhibits spontaneous

exudation of oil. All diatoms tested by us produced oil but the visible oil content seemed to be

maximum in Diadesmis confervacea, which was proved by transesterification. Diatoms with high lipid

content and naturally oozing would eliminate the expensive step in energy production of extracting oil

out of the diatoms [29]. They may prove to be key elements towards our goal of constructing biofuel

generating diatom solar panels [7].

The water body of Haryana, India that we selected, known as Sultanpur Lake, has slightly high pH

(7.88) and conductivity (0.42 S/m) during autumns compared to other seasons of the year. Commonly

occurring diatoms in Sultanpur Lake were Achnanthes, Cymbella, Cocconeis, Navicula, Gomphonema,

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Nitzschia, Encyonema and Cyclotella. Besides these some rarely occurring diatoms were Diadesmis,

Reimeria, Sellaphora, Surirella, Luticola, Pinnularia, Melosira, Opephora, Synedra and Placoneis

(Plate 1). Pure diatom cultures of Diadesmis confervacea Kützing, Nitzschia palea (Kützing) W.

Smith, Cyclotella menenghiana Kützing, Pinnularia borelias (Ehrenberg) Rabenhorst, Placoneis

elginensis (Gregory) E.J. Cox and some new species of Nitzschia were obtained from this natural water

body. Among these pure diatom cultures of D. confervacea (Plate 2) was selected due to its maximum

oil content of 14.6% as compared, for example, to 5% in Nitzschia palea.

2. Results and discussions

In the present study about twelve pure cultures of diatom from fresh water bodies were obtained which

included Diadesmis confervacea Kützing, Nitzschia palea (Kützing) W. Smith, Cyclotella

menenghiana Kützing, Pinnularia borelias (Ehrenberg) Rabenhorst, Placoneis elginensis (Gregory)

E.J. Cox and some new species of Nitzschia. D. confervacea proved to have both high lipid content

and natural, spontaneous secretion of its oil. D. confervacea had the highest lipid content about 14.6%

as compared to Nitzschia 5% and thus was selected as the appropriate candidate for formation of

diatom solar panels. The in vivo growth of this diatom on modified f/2 media showed that 50% of the

cells oozed oil at 10th day of inoculation and 80% of the cells oozed oil spontaneously on 31st day of

inoculation. The axenic diatom culture was scraped from the culture media plate and transferred into

100 µl of freshly prepared chloroform: methanol (1:1) solution in a vial followed by transesterification

of oil in diatoms to fatty acids methyl esters and glycerol [30]. The TLC (thin layer chromatography)

chromatogram shows separation at different retention factors (Rf) which is defined as the distance

traveled by the compound divided by the distance traveled by the solvent. Rf for different components

was viz. cholesterol ester (Rf=0.88), triacyl glycerol (Rf=0.52), free fatty acids (Rf=0.45, 0.40 and

0.107), 1,3 diacyl glycerol (Rf=0.083), 1,2 diacyl glycerol (Rf=0.0357) and monoglycerols

(Rf=0.0238) for the transesterified sample in quadruplets (Figure 6). The GCMS results showed that

transesterification has resulted in formation of free fatty acids at retention time peak near C-12.91 and

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some methyl esters (FAME) at retention time peak near C-18.32 (Figure 7 and Table 1), besides this

few other small peaks showing formation of esters and presence of fatty acids in a solvent mixture of

chloroform: methanol (1:1) were seen at retention time of approximately 30 minutes. One peculiarity

with D. confervacea is that the cells increased in size during the course of the experiment, rather than

decreased. This increase was not great in the valve to valve distance (Figure 2), suggesting that the size

increase is not due to an increased number of girdle bands. Rather, the tip to tip length of the valves

increased (Figure 3). Some diatom species do not show the usual decrease in size of the cell during cell

division [30].

It is worth considering some consistencies and inconsistencies in our data. The valves for which we

measured W and S to get their ratio have a larger value of W than that in Figure 5. Perhaps individual

cells teased out of these colonies are buckled [31] under tension and change shape.

The weight of transesterified lipid and average diatom cell count per 100 µl from 1st day to 31st day of

diatom D. confervacea was recorded which showed increase in FAME at 9th day and 24th day, however

oil oozed at around 31st day. The diatom count also raised significantly it being maximum between 13th

and 27th day thereafter growth retarded and oil started occupying whole volume of the diatom cell

(Figure 6). The oil in the diatoms expanded at the later stages and took the shape of the cell as a whole

(Plate 4 and 5). At 31st day onwards the oil started oozing from the diatom cells naturally without any

force, mechanical pressure or heat and was left with light brown chloroplast which again divided and

produced oil when fed with fresh nutrient media (Plate 6 & 7). Thus growth of diatoms especially D.

confervacea in in vivo conditions resulted in oozing of oil without cell lysis making it as good

candidate for construction of diatom solar panels.

The concept of milking oil from diatoms [7] can be achieved in a simple way: oozing of oil after one

month suggests that the nutrient medium is now deficient, perhaps providing the stress that increases

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oil production without having to prepare media deficient in nitrates or silicates [18]. When supplied

with fresh nutrient media the diatom cells can ooze oil again.

Our results suggest that extraction of diatom oil using hydrocarbons [32, 33] or other means [34] is not

necessary, and that the oil comes out spontaneously from some diatoms without damage to the cells.

Furthermore, additional energy beyond that taken up by photosynthesis need not be supplied, nor is

genetic engineering [29, 35] necessary for oil exocytosis. Cultures that exuded their oil are capable of

doing it again, meeting a primary requirement for the potential success of diatom solar panels [7].

3. Methods

3.1 Taxonomy and Culturing

Diatom taxa were identified using various keys [36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50]. The water sample brought into the laboratory was studied first for its species richness and later on

was inoculated onto the prepared culture media. Each aliquot was cultured on f/2 solid agar

medium [51] with the following modifications: soil extract (5ml/liter), Na2SiO3.5H2O at a

concentration of 40 g/l (2ml/liter), with the final volume of medium made up to 1 liter by adding

autoclaved double distilled water. Brown diatom colonies appeared when grown at 18 ± 1°C under

18000 lux, light source: white/yellow light (25W tungsten bulb), light: dark conditions: 16 hrs/8 hrs.

The mixed colonies of cultured diatoms were then serial diluted to obtain axenic cultures. Possible

bacterial contamination, in case any remained, was treated with a mixture of Penicillin G (170 µg/l)

and Streptomycin (85 µg/l) in the ratio of 1:0.5 using a Metler Toledo AB 265-S/ FACT weighing

balance [52]. 100 µl of pure colonies of diatom culture at 6800cells/100µl were inoculated onto each

of 31 Petri plates of diameter 7.7 cm, labeled as 1st day to 31st day:

1. Each day the respective plate was observed for the presence and size of oil globules in the

diatom cells.

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2. The exocytosis of oil from the diatom cells was also looked for on each day after their

inoculation.

3. The number of cells was counted on each day.

4. Each day the corresponding plate was also subjected to a transesterification process to estimate

the oil content [53].

5. Fatty acid methyl esters (FAME) were analyzed by GCMS and tested using TLC.

3.1.1 Morphological Study:

All the observations, length/breadth or diameters of individual oil droplets in duplicate were averaged

over usually five diatom cells in a single image recorded at 100X observation using a Leica DM 2500

compound microscope [54]fitted with Leica EC3 digital camera and using SEM Zess EVO MA

10(Plate 3) . Cells were observed in situ, on the medium in the Petri dishes, without the use of a cover

slip, which otherwise might mechanically cause oil expression [55]. The volumes of individual oil

droplets and whole diatom cells were determined by assessing the geometric shape of the cell or oil

globule [56]. Generally each oil globule had the projected shape of a circle or a capped rectangle. Clear

images with no overlapping of the filamentous diatom chains were selected for the measurement and

their sizes were measured using LAS EZ software [57].

The weight of the lipid content from 1st day till 31st day of inoculation was measured by the

transesterification process (Figure 1).

Figures 2 and 3 show that as the diatoms grow on the enriched medium their numbers not only

increase approximately exponentially but the cell size also increases. The oil content also peaks as the

culture matures. The diatom cells seem to finally exude oil when the cell size no longer increases in

size but the oil content inside the cell increases its volume until it finds no space inside and bursts open

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the cell indicating there is a correlation between a morphological pattern of diatom growth and its oil

content. During the log or exponential phase during first 15-20 days of growth as shown in Figures 2

and 3, the lipid content took the shape of the cell, at least in girdle view (Plate 4 and 5). There were

small fluctuations in cell and lipid volume as all cells are not morphologically of the same size and

shape.

3.1.2 Volumetric Analysis:

A fixed volume (100 µl) of D. confervacea was inoculated onto each of 31 media Petri plates.

Colonies with maximum oil content were selected systematically, a form of taking the sample

maximum in statistics [58]. The following measurements were made for each selected cell in a colony:

L = length of cell along the axis of the colony = distance between valves of a cell

W = width of cell perpendicular to the axis of the colony

d = diameter of each spherical oil droplet in the cell

We calculated the oil droplet volume using the formula of Archimedes:

v = 43π d2

⎛⎝⎜

⎞⎠⎟3

For nonspherical oil droplets we approximated the shape as a cylinder with spherical caps and

measured:

l = longest straight side

w = shortest side (diameter of cylinder)

and estimated the volume as:

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v = π w2

⎛⎝⎜

⎞⎠⎟2

l + 43π w

2⎛⎝⎜

⎞⎠⎟3

One parameter was not measured daily because of the orientation of the colonies:

S = maximum width of the valve

From the live cells in Plate 1 and 2 that are seen in valve view we estimate that W/S = 2.1 and that we

can approximate the valve surface area as an ellipse. This allows us to estimate the volume of the cell

as:

V = π W2

⎛⎝⎜

⎞⎠⎟S2

⎛⎝⎜

⎞⎠⎟ =

π4WS

or, approximatingW ≈ 2S :

V ≈ π8W 2

Figures 2 and 3 depict V and v and Figures 4 and 5 depict L and W versus each day, including standard

deviations. Plate 4 and 5 show the exocytosis pattern from diatom cells and their growth pattern over

one month duration. The average volume of oil droplets in the diatom cells was 35 µm3, initially, at the

time of inoculation. The diatoms started showing visible growth on the 4th day.

Figures 2-5 show large fluctuations that may be due to stochastic variations in the independent growth

in each Petri dish.

Plates 4 and 5 show exudation of oil from diatom cells on the 31st day, and the color change in

pigmentation is observed changing from golden brown to slightly pale. This is due to the exudation of

the oil content which when inside the cell pushes the chloroplast content together, which appears dark

golden brown; but once the oil exudates, the chloroplast spreads in the cell. After the 31st day D.

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confervacea was subcultured on fresh culture media plates and after 14 days revived colonies were

obtained containing oil droplets as shown in Plate 6. The lipid content present within the revived

colonies of D. confervacea was stained by Sudan-IV dye [59] as shown in Plate 7.

Acknowledgments

We would also like to thank Karthick Balasubramanian and Kalina M. Manoylov who kindly assisted

with identification of Diadesmis confervacea, and we thank KMM for a critical reading of the

manuscript. We would also like to thank the Department of Biotechnology Government of India for

financial and laboratory support, and the Gulf Specimen Marine Laboratory and Birds Hill Provincial

Park for congenial natural environments for writing.

Conflicts of Interest

The authors declare no conflict of interest.

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58. Wikipedia Sample maximum and minimum. Available online: http://en.wikipedia.org/wiki/Sample_maximum

59. Crawford, R.M. The protoplasmic ultrastructure of the vegetative cell of Melosira varians C.A. Agardh. J. Phycol. 1973, 9, 50-61.

14

5. Figure Legends:

Figure 1. Average volume V of diatom cells in µm3 and its standard error for samples of 5 Diadesmis

confervacea diatoms on a single plate, 2 plates per day, i.e., 10 cells measured per day. The day refers

to the end of each 24 hour period, i.e., plate’s labeled day 1 were measured 24 hours after inoculation.

The black bars show one standard error above the mean (top of blue bars).

Figure 2. Average volume v of oil per droplet in µm3 and its standard error for samples of Diadesmis

confervacea diatoms on a single plate, 2 plates per day. The day refers to the end of each 24 hour

period, i.e., plate’s labeled day 1 were measured 24 hours after inoculation. Five diatom oil droplets

were measured per plate, i.e., 10 oil droplets per day.

Figure 3. Average valve to valve length L of diatom cells in µm, measured along the axis of the

colony, and its standard error for samples of 5 Diadesmis confervacea diatoms on a single plate,

2 plates per day, i.e., 10 cells measured per day. The day refers to the end of each 24 hour period,

i.e., plate’s labeled day 1 were measured 24 hours after inoculation. The black bars show one

standard error above the mean (top of blue bars).

Figure 4. Average width W of diatom cells in µm, measured perpendicular to the axis of the

colony (i.e., length of the long axis of the valve), and its standard error for samples of Diadesmis

confervacea diatoms on a single plate, 2 plates per day, i.e., 10 cells measured per day. The day

refers to the end of each 24 hour period, i.e., plate’s labeled day 1 were measured 24 hours after

inoculation. The black bars show one standard error above the mean (top of blue bars).

Figure 5. Weight of transesterified lipid in micrograms (µg) and average diatom cell count per 100µl

from 1st day to 31st day of diatom Diadesmis confervacea. The day refers to the end of 24 hours, i.e.,

plate labeled day 1, were counted and transesterified 24 hours after inoculation. The red bars show

weight of transesterified lipid (µg) and green bars show average diatom cell count of Diadesmis

confervacea per 100 µl.

15

Figure 6: TLC of the oil extracted from Diadesmis confervacea (T) showing separation of cholesterol

ester (Rf= 0.88), free fatty acids (FFA) (Rf=0.45, 0.40 and 0.107), 1,3 diacyl glycerol (1,3 DAG)

(Rf=0.083), 1,2 diacyl glycerol (1,2 DAG) (Rf=0.0357) and monoglycerols (MG) (Rf=0.0238) with

standards- S1: Mustard oil, S2: Olive oil, S3: Cocconut oil, S4: Soyabean oil and S5: Codliver oil.

Figure 7 Chromatogram of oil extracted from Diadesmis confervacea analyzed in GCMS for first

(tR=Retention time) 19 minutes in which maximum free fatty acids and FAME were detected.

Plate 1. Microscope images of few diatoms containing lipid drops from the water sample of

Sultanpur Lake.

Plate 2. Microscope images of pure diatom cultures of Diadesmis confervacea (Ehrenberg)

Mereschkowsky.

Plate 3. SEM images of Diadesmis confervacea

Plate 4. Compound microscope images of Diadesmis confervacea diatoms showing the lipid

content growth pattern and exocytose of oil over one month. Each image is of a small sample

taken from the first culture media plate for that day (in duplicate). Lines are drawn by hand using

LAS EZ software [57] to indicate the measurements of oil droplets in the following manner:

Red lines indicate (------) Diameter of sphere = d

Pink line indicate (------) Diameter of cylinder = w

Blue line indicate (------) Length of cylinder = l

Plate 5. Compound microscope images of Diadesmis gallica diatoms and image is of a small sample

taken from second culture media plate for that day(in duplicate), with their whole diatom cell

measurement. Lines are drawn by hand to indicate the measurements in following manner:

Pink lines indicate (------) Width of cell perpendicular to the axis of the colony = W

Blue lines indicate (------) Length of cell along the axis of the colony = distance between valves of a

cell = L

Plate 6. Microscope images of revived colony of diatom which was subculture after 31st day and

observed on 14th day.

Plate 7. Sudan IV staining of lipid droplets inside the diatom cell (Diadesmis confervacea) of

revived colonies.

16

Figure 1. Average volume V of diatom cells in µm3 and its standard error for samples of 5 Diadesmis

confervacea diatoms on a single plate, 2 plates per day, i.e., 10 cells measured per day. The day refers

to the end of each 24 hour period, i.e., plate’s labeled day 1 were measured 24 hours after inoculation.

The black bars show one standard error above the mean (top of blue bars).

Figure 2. Average volume v of oil per droplet in µm3 and its standard error for samples of Diadesmis

confervacea diatoms on a single plate, 2 plates per day. The day refers to the end of each 24 hour

period, i.e., plate’s labeled day 1 were measured 24 hours after inoculation. Five diatom oil droplets

were measured per plate, i.e., 10 oil droplets per day.

17

Figure 3. Average valve to valve length L of diatom cells in µm, measured along the axis of the

colony, and its standard error for samples of 5 Diadesmis confervacea diatoms on a single plate,

2 plates per day, i.e., 10 cells measured per day. The day refers to the end of each 24 hour period,

i.e., plate’s labeled day 1 were measured 24 hours after inoculation. The black bars show one

standard error above the mean (top of blue bars).

Figure 4. Average width W of diatom cells in µm, measured perpendicular to the axis of the

colony (i.e., length of the long axis of the valve), and its standard error for samples of Diadesmis

confervacea diatoms on a single plate, 2 plates per day, i.e., 10 cells measured per day. The day

refers to the end of each 24 hour period, i.e., plate’s labeled day 1 were measured 24 hours after

inoculation. The black bars show one standard error above the mean (top of blue bars).

18

Figure 5. Weight of transesterified lipid in micrograms (µg) and average diatom cell count per

100µl from 1st day to 31st day of diatom Diadesmis confervacea. The day refers to the end of 24

hours, i.e., plate labeled day 1, were counted and transesterified 24 hours after inoculation. The

red bars show weight of transesterified lipid (µg) and green bars show average diatom cell count

of Diadesmis confervacea per 100 µl.

Figure 6: TLC of the oil extracted from Diadesmis confervacea (T) showing separation of cholesterol

ester (Rf= 0.88), free fatty acids (FFA) (Rf=0.45, 0.40 and 0.107), 1,3 diacyl glycerol (1,3 DAG)

(Rf=0.083), 1,2 diacyl glycerol (1,2 DAG) (Rf=0.0357) and monoglycerols (MG) (Rf=0.0238) with

standards- S1: Mustard oil, S2: Olive oil, S3: Cocconut oil, S4: Soyabean oil and S5: Codliver oil.

Chlolesterol ester

Free fatty acids

1,3 DAG

1,2 DAG

MG

Figure 7 Chromatogram of oil extracted from Diadesmis confervacea analyzed in GCMS for first

(tR=Retention time) 19 minutes in which maximum free fatty acids and FAME were detected.

19

Plate 1. Microscope images of few diatoms containing lipid drops from the water sample of

Sultanpur Lake.

20

Image 1: Cyclotella meneghiniana Image 2: Nitzschia palea

Image 3: Pinnularia borealis Image 4: Diadesmis confervacea

21

Plate 2. Microscope images of pure diatom cultures of Diadesmis confervacea (Ehrenberg)

Mereschkowsky.

Image 1: Colony of Diadesmis confervacea (10x)

on solid agar culture medium plate.

Image 2: Pure culture of Diadesmis confervacea

(100x) and its filamentous form in girdle view.

Image 3: Solitary cells of Diadesmis confervacea

(100x) after teasing.

Image 4: Solitary cells of Diadesmis confervacea

(100x) after drying and mounting in DPX.

22

Plate 3. SEM images of Diadesmis confervacea

Plate 4. Compound microscope images of Diadesmis confervacea diatoms showing the lipid

content growth pattern and exocytose of oil over one month. Each image is of a small sample

23

taken from the first culture media plate for that day (in duplicate). Lines are drawn by hand using

LAS EZ software [57] to indicate the measurements of oil droplets in the following manner:

Red lines indicate (------) Diameter of sphere = d

Pink line indicate (------) Diameter of cylinder = w

Blue line indicate (------) Length of cylinder = l

1st Day(i) 1st Day (ii)

10th Day(i)

10th Day(ii)

24

29th Day(i)

29th Day(ii)

31st Day(i)

31st Day(ii)

Plate 5. Compound microscope images of Diadesmis gallica diatoms and image is of a small sample taken

from second culture media plate for that day(in duplicate), with their whole diatom cell measurement. Lines

are drawn by hand to indicate the measurements in following manner:

Pink lines indicate (------) Width of cell perpendicular to the axis of the colony = W

25

Blue lines indicate (------) Length of cell along the axis of the colony = distance between valves of a cell

= L

1st Day(i) 1st Day (ii)

10th Day(i)

10th Day(ii)

26

29th Day(i)

29th Day(ii)

31st Day(i)

31st Day(ii)

Plate 6. Microscope images of revived colony of diatom which was subculture after 31st day and

observed on 14th day.

27

Revived colony of diatom (Diadesmis

confervacea) of

Revived colony of diatom (Diadesmis

confervacea)

Plate 7. Sudan IV staining of lipid droplets inside the diatom cell (Diadesmis confervacea) of

revived colonies.

Revived colony of diatom (Diadesmis

confervacea) stained with Sudan IV.

Revived colony of diatom (Diadesmis

confervacea) stained with Sudan IV.

28

Table 1: GCMS results of the FAME of retention time peak at C-18.32.

Peak-18.32 Compound Name M.W. Formula

Cholest-5-ene,3-Bromo-,(3-Beta)- 448 C27H45Br Cholest-5-en-3-ol (3-Beta)-, Carbonochlo 448 C28H45O2Cl Cholest-5-on-3-ol (3-Beta)-,Acetate 526 C36H62O2 Cholesteryl Benzoate 428 C29H48O2 Cholesta-3,5-diene 490 C34H50O2 Cholesterol 368 C27H44 Cholesta-3,5-diene 386 C27H46O Cholest-5-en-3-ol (3-Beta)-Propanoate 368 C27H44 Cholestane-3,5-diol, diacetate (3-Beta), 5.A 442 C30H50O2 Cholest-5-en-3-ol (3-Beta)-, Tetradecanoa 488 C31H52O4 Cholesta-3,5-diene 596 C41H72O2 Cholest-5-en-3-ol (3-Beta)-,3-Phenyl-2-PR 368 C27H44 26,27-Dinorergost-5-en-3-ol, Benzoate, 516 C36H52O2 Cholesteryl Butyrate 476 C33H48O2 Cholesteryl Benzoate 456 C31H52O2 Cholestadiene 490 C34H50O2 Beta-Sitosterol Acetate 368 C27H44 Stigmastan-3-ol,5-Chloro-Acetate, (3.Beta) 456 C31H52O2 Methyl(25RS)-3.Beta-Acetoxy-5-Choleste 492 C31H53O2Cl