Endolithic Cyanobacterium, Leptolyngbya ISTCY101, for Synergistic Biofuel Production

and Wastewater Treatment

Presented By

Jyoti Singh

School of Environmental SciencesJawaharlal Nehru University

New Delhi-110067

Biggest challenges Human race is facing

Exhausting fossil fuels Climate change (Global warming)

The most troublesome Green House Gas

BACKGROUND INFORMATION

3726 kg water is required to generate 1 kg microalgae biodiesel

energy & fertilizer consumption, most economically cumbersome

higher environmental burden in terms of energy , water consumption & GHG emissions

harness solar energy and transform CO2

into fuels and other valuable bioproducts

live in a wide spectrum of environments and have minimal nutritional requirement

don’t require arable land

Have appreciable lipid content 10-70 %

What becomes important now…. Isolating and screening of potential microalgae from unexplored sources is an indispensable research area

Integrating an already existing system with microalgal biofuel production for better sustainability

PLAN OF WORK

Sampling, Isolation and

Identification of cyanobacteria

Characterization of growth attributes

and biomass productivity

Wastewater sampling and

characterization

Wastewater treatment using cyanobacteria

Lipid estimation and fatty acid

profiling

1

2

3

45

1 2

3

45

Cyanobacterial samples

Inoculation in BG11 medium + NaHCO3

Most competitive strain

16s rDNA sequencing

Strain identification (NCBI BLAST)

Identified Cyanobacterial strain

Cultures maintained at differentpHs

TemperatureSalinity

Growth determination (as a function of

Biomass)

Sewage Wastewater

Temperature, pH, BOD, COD,

nitrate, ammonia,

Phosphorus, cations,

Total organics

Isolated Cyanobacteria

Inoculation in sterilized waste water for 14 days

Biomass Heavy metal determination, total organics, genotoxicity evaluation

Growth determination & Lipid extraction

Biomass

Modified Bligh-Dyer extraction

Transesterification with KOH

FAMEs (GCMS analysis)

METHODOLOGY

SAMPLING ISOLATION AND IDENTIFICATION OF MICROALGAE

Cyanobacterial samples collected from Jhiri marble mining site

Microlagae growing in the fissures and pores of marble rock were scrapped

Inoculated into conical flasks with 500ml modified BG11 medium (10 mM, 50 mM, 100mM NaHCO3)

Best growing strain

16S rDNA PCR amplification using the primer pair CYAN 106F/738R

OD at 750nm

DNA isolation from axenic culture

CHARACTERIZATION OF GROWTH ATTRIBUTES AND BIOMASS PRODUCTIVITY

Isolated strain

Temperature10°C, 20°C, 30°C,

40°CGrowth determined [function of biomass

production per day (g/L)]. 10mL of sample

centrifuged at 8000 g for 15 min. supernatant added back into culture medium and microalgal biomass

oven dried at 100°C for 8 hrs.

NaCl25 g/L & 35 g/L

pH7, 8, 9, 10, 11

Wastewater Sampling and Characterization

Municipal Sewage Water Treatment PlantVasant Vihar, New Delhi

Physicochemical parameters (temperature, pH, BOD, COD, nitrate, ammonia and total

phosphorus)

Major elements and heavy metals were analysed using ICP-OES in compliance with US EPA

method 200.7

Total organics were extracted using 3 volumes of DCM:

Acetone solvent mixture (1:1 v/v) and analysed by GCMS

Samples from influent tank were collected as per the guidelines given in Standard Methods for the Examination of Water and

Wastewater, APHA 2005

Wastewater treatment using Cyanobacteria

Strain ISTCY101 inoculated into conical flasks with 1L filter sterilized wastewater (25%, 50%, 75%, 100%) strictly as growth medium under optimized culture

conditions for 14 days

cells were harvested, total lipids were extracted, transesterified and analyzed by GC-MS

Removal of heavy metals and organic pollutants was determined via ICP-OES and GC-MS respectively

Lipid analysis and fatty acid profileDried algal samples (1g) [cell disruption in a 130W sonicator for 20 min & extracted for 30 min with 30 mL of chloroform/methanol (2:1 v/v)

separated out different layers. lipid fraction was separated and the solvent evaporated using a vacuum rotary evaporator.

Transesterification reaction (oil: methanol molar ratio 1:6, KOH (1%), incubated for 30 min at room temperature).

Resulting FAMEs were recovered into 1 ml of chloroform. The chloroform layer was clarified by centrifugation and sampled for GCMS analysis.

FAMEs identification via data comparison with the inbuilt standard mass spectra library system (NIST-05 and Wiley-8) of GC–MS

RESULTS

Days

0 3 6 9 12 15 18

OD

(750

nm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

ISTCY101 CY 2 CY 3 CY 4 CY 5CY 6CY 7

•Seven microalgal strains namely ISTCY101, CY2, CY3, CY4, CY5, CY6, CY7 were isolated from marble rock

•ISTCY101 grew well even at 100mM sodium bicarbonate concentration

•Though CY2 and CY3 endured higher sodium bicarbonate concentration, ISTCY101 came out to be best

Growth curves of 7 isolated strains inoculated in BG 11 medium amended with sodium bicarbonate

(50mM)

•Based on 16S rRNA data, isolated strain was identified as Leptolyngbya sp. ISTCY101 (Accession no. KC538900) and may be considered novel. BLAST results showed maximum similarity to species belonging to genera Leptolyngbya.

•Leptolyngbya sp. ISTCY101 belongs to a group of oscillatorians (Fig. 2), mostly belonging to the genus Leptolyngbya, including strain Leptolyngbya sp. MMG-1 and Leptolyngbya sp. Greenland 7.

•Leptolyngbya is one of the most reported genera found thriving on dolomite and other carbonate rocks, spanning the outer millimetre to inner centimetres of rocks exposed to extreme environments

Phylogenetic tree showing the relationships among rDNA sequences of isolate ISTCY101 and the most similar

sequences retrieved from the NCBI nucleotide database.

Medium Specific Growth Rate (fw day-1)

Biomass Productivity DW(mg/L/day)

Lipid productivity(% wt of total dry wt)

BG11 10.55 62.01 200mg/gArtificial Sea Water

25g NaCl/L35g NaCl/L

9.889.51

60.9360.21

210mg/g160mg/g

BG11+NaHCO3

10mM50mM

100mM

10.611.89.46

6470.2359.1

190mg/g200mg/g180mg/g

pH789

1011

++++++++++++++

Temperature °C+

+++++++

10203040

Growth attributes of Leptolyngbya ISTCY101

Days

0 2 4 6 8 10 12 14 16

Bio

mas

s (g

/L)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Bicarbonate 50mMBG11Wastewater (100%)

Biomass yield in different growth media

•The biomass productivity of Leptolyngbya ISTCY101 under optimum conditions was recorded to be 70.23 mg/L/day

•In undiluted wastewater, Leptolyngbya ISTCY101 exhibited an average dry biomass productivity of 85.45 mg/L/day

Temperature (°C) 18.6 ± 0.5pH 7.2 ± 0.3

Electrical Conductivity (mS) 1.8 ± 2.1

TDS (ppt) 0.9 ± 1.1BOD5(mg l–1 O2) 350 ± 3.5COD (mg l–1 O2) 720 ± 12.4

Nitrate 0.261±0.03Ammonium 21.6 ± 2.3

Total Phosphorus 27.1± 4.1

Physicochemical parameters of wastewater

The primary characteristics in terms of COD, BOD, TDS, and major nutrients were found to be moderate and comparable to avg. values found in domestic sewage

wastewater.

Cations (mg/L) Before Treatment After TreatmentAl 72.34 33.25Cd 0.02 Not detectedCo 0.02 Not detectedFe 0.52 Not detectedK 34.84 28.74

Mg 23.09 8.95Na 145.37 136.52Ni 0.03 Not detectedPb 0.02 Not detectedZn 0.69 0.51

Major elements present in wastewater

•Among the major elements detected, concentration of Al was particularly found to be high i.e. 72.34 mg/L

•Heavy metals i.e. Cd, Pb, Ni, Co were also detected. 60-70% of load of Cd, Zn, Cu, and Ni in domestic waste water is contributed by faeces. Other prime sources are pharmaceuticals, cleansers, detergents, cosmetics and body care products

•Al was reduced by more than 54%, whereas Cd, Co, Fe, Pb, Ni were not detected post treatment. Concentrations of K, Na, Mg and Zn were also reduced markedly

Pretreatment Post treatment

SEM micrographs depicting the morphological changes seen in Leptolyngbya ISTCY101 after 14 days treatment of wastewater

2 4 6 8 10 12 14 16 18 20keV

0

2

4

6

8

10

cps/eV

C O

Na Mg

Al

Ca

Ca

Cl

Cl

2 4 6 8 10 12 14 16 18 20keV

0

2

4

6

8

10

12

14

16

18

20

22

cps/eV

C O

Si Ca

Ca

Na Cd Cd

Cd

Ni Ni

Cr

Cr

Pb Pb Pb Fe

Fe

Mg Al

•The metal-binding properties of phototrophic biofilms can be attributed to the presence of extracellular polysaccharides that are negatively charged at high pH levels generated by oxygenic photosynthesis.

•Besides biosorption and bioaccumulation, the increased pH inside photoautotrophic biofilms may cause metals removal by precipitation

•SEM EDX results confirmed the presence of Cadmium, Nickel, Chromium, Lead, Iron on the cell surface of Leptolyngbya ISTCY101

Benzoic acid, 4-ethoxy-, ethyl ester

Squalene

Oleic Acid

Octadecanoic acid

BENZENE, (1-PENTYLOCTYL)-

Cholestane-3,5-diol, 5-acetate, (3.beta.,5.alpha.)-CHOLESTAN-3-OL, (3.BETA.,5.ALPHA.)-

SILICONE OIL

beta.-Sitosterol

1,2-BENZENEDICARBOXYLIC ACID, DIETHYL ESTER

PHENOL, 2,6-BIS(1,1-DIMETHYLETHYL)-

PHENOL, 4-CHLORO-3,5-DIMETHYL-

PHENOL, 4-METHYL-

PHENOL, 2-METHOXY-3-(2-PROPENYL)-

9-OCTADECENOIC ACID

METHYL (3-OXO-2-PENTYLCYCLOPENTYL)ACETATE

CHOLESTAN-3-OL, (3.BETA.,5.ALPHA.)-

•Major organic constituents found after GC-MS analysis were fatty acids mainly Oleic Acid and Octadecaenoic acid

•Phenol and benzene derivatives were also detected including phenol 4-methyl, benzene (1-Pentyloctyl). Some sterols including beta-sitosterol were also detected

•The large presence of fatty acids in domestic waste water can be attributed to the regular use of detergents, soaps and cosmetics in households.

Retention time Name6.305 3-cyclohexene-1-methanol8.960 Trimethylsilyl palmitate9.857 Trimethylsilyl palmitate

10.311 9-octadecenoic acid, methyl ester11.286 Tetratriacontane11.909 1,2-benzenedicarboxylic acid12.683 Nonacosane15.341 4'-biphenylene diphosphonate

Organic compounds detected in wastewater after treatment

•Phthalic acid and 3-cyclohexene-1-methanol were the major compounds detected after cyanobacterial treatment of wastewater, rest of the proportion was contributed by fatty acid derivatives

•Many studies have reported the efficiency of Cyanobacteria to accumulate and degrade a vast range of environmental pollutants, including pesticides, crude oil, Polycyclic Aromatic Hydrocarbons, phenol and catechol and xenobiotics.

•The presence of phthalic acid can be attributed to the leaching of phthalates from plasticwares used during processing as well as microbial degradation of organic compounds

Results of the comet assay for untreated and treated wastewater samples. The comets were divided into five classes on the basis of amount of DNA in tail. HepG2 cells treated with the untreated (UT) sample resulted in 100% comets that fell under classes IV and V. Whereas, in Treated sample, percentage of comets lying in class V was reduced to 0 and more than 90%

comets lied in class II.

V I IV II

BaP 50 µM MQ 0.5% Untreated (UT) Treated

Alkaline single-cell gel electrophoresis (Comet assay) for toxicological evaluation of wastewater

FAMEs

Per

cent

age

yiel

d

0

20

40

60

80

100

120

Palmitic acidPalmitoleic acidHexadecadienoic acidHexaecatrienoic acidDocosahexaenoic AcidStearic acidOleic acidLinoleic acidLinolenic acidCis,cis Linoleic acid

•Fatty acid profiles analyzed by GC-MS show 96% FAMEs (Fatty Acid Methyl Esters) yield

•Higher proportion of Linoleic acid (30%), Oleic acid (17%) and Palmitic acid (19.06%)

•In particular, oils with high oleic acid content have been reported to have a reasonable balance of fuel properties

•The other important FAME that was detected is Docosahexaenoic Acid (DHA) (3.46%) also known as cervonic acid

•The proportion of FAMEs in lipid extracted from Leptolyngbya ISTCY101 is balanced in terms of saturated and unsaturated fatty acids which make it suitable to produce a good quality biodiesel

Fatty acid profile of leptolyngbya ISTCY101 grown on wastewater as growth medium

Conclusions•The isolated strain Leptolyngbya ISTCY101 is a potential candidate for cyanobacterial research dedicated to alternative energy.

•Leptolyngbya ISTCY101 exhibited robust growth in undiluted wastewater, with a biomass productivity of 85.45 mg/L/day and 20% lipid yield per unit dry wt as compared to 70.23 mg/L/day in BG11 medium (50 mM NaHCO3).

•Furthermore, it accumulated high FAME content i.e. 96% of its total lipid, which is higher as compared to usual microalgal feedstock for biodiesel production.

•Leptolyngbya ISTCY101 proficiently used wastewater for synergistic production of biodiesel and removal of major contaminants, thus providing a cost effective alternative approach for mass scale bioenergy production and primary wastewater treatment.

•The strain can be subjected to further genetic improvements to provide an efficient alternative approach to recycle CO2 into fuels and other bioproducts

•Furthermore, the study opens up new avenues like marble mines for exploring microorganisms with enormous potencies for use in CO2 recycling, biofuel/bioproduct production and bioremediation

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