Institute of Chemical Technology

Presentation at National Conference on VBBBNA2018 Madras Christian College, Tambaram, Chennai September 20-22, 2018 ICT-Mumbai

An invited talk

C R K Reddy DBT Energy Biosciences Chair DBT-ICT Centre for Energy Biosciences

Institute of Chemical Technology Matunga East, Mumbai 400 019

Composition of different macroalgal species ICT-Mumbai

(Source: Prof. John Bothwell, Durham University, Durham)

Overview of Presentation

 International Scenario  National Status  Road map for making accelerated growth A) Technical  Efficient cultivation methods  Abiotic factors  Farm management  Post harvesting strategies B) Scientific  Domestication  Improved and resilient strains  Nursery development  Environmental impact  Application of advanced knowledge  Downstream Process  Scale up process  Value addition  Green process  Seaweed Policy  Seaweed development board

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• World population is increasing by 35% while India’s population increases by 41% • According to FAO, world need 70% (5 billion MT) more food production by 2050, while India needs 80% more

Marine resource potential massive !!!!

• The oceans cover 70% of earth yet they yield only 1.2% (120 million metric tons (MT) of the 10 billion MT of food that produced each year

• Farmed seaweed production 99.4% from seven countries in Asia (FAO) China, Indonesia, Philippines, Korea, Japan, Malaysia and Vietnam

Global mcroalgal diversity ICT-Mumbai

Rhodophyta

• ≈ 6500 species worldwide • 434 species from India

Phaeophyta

• ≈ 1800 species worldwide • 194 species from India

Chlorophyta

• ≈1500 species worldwide • 216 species from India

Current production estimated at 31.2 million tons wet wt. with USD 11.7 billion FAO 2018

Source: http://www.seaweed.ie/

There are about 10,000 seaweed species reported worldwide

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World map showing the status of seaweed farming

Current production estimated at 31.2 million tons wet wt.

Red: Intense seaweed farming; Green: moderate seaweed farming; Yellow colour: no seaweed farming

Laminaria japonica (Kombu) farm - China ICT-Mumbai

Photo courtesy Prof. Chen Jiaxin

Species-wise production data Value US$ 11.7 billion

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Sargassum fusiforme 1% Others 1% Undaria pinnatifida 9% Eucheuma spp 35%

Laminaria japonica 29%

Gracilaria spp. 14% Porphyra spp. 7%

Kappaphycus alvarezii 6%

Current production estimated at 31.2 million tons wet wt. with FAO 2018

Extrapolated ecosystem services from 500 million tons (dry weight) of seaweeds ICT-Mumbai

Seaweed Aquaculture for Food Security, Income Generation and Environmental Health, World Bank Group.

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Places of algal research in India Marine facts

a

Delhi

Continental shelf

372, 424 sq. km

EEZ

2,103,415 sq. km

Coastline

7500 km

Total seaweed flora

840 sppa

Total standing stock

600,000 tons fresh wt.

Oza and Zaidi 2001

Gujarat

West Bengal Maharashtra

Lakshadweep Islands

Goa Karnataka

Orissa

Andhra Pradesh A&N Islands

Kerala

Seaweed beds in nature

Tamil Nadu

Economically important seaweeds of India ICT-Mumbai

Phycocolloid seaweeds Edible seaweeds

Gracilaria dura

Ulva

Gracilaria edulis

Monostroma

Caulerpa lentillifera

Kappaphycus alvarezii

Porphyra

Gelidiella acerosa

Gracilaria verrucosa

Sargassum tenerrimum

Turbinaria conoides

List of granted/filed patents on seaweeds: CSIR-CSMCRI

Sr. No 1

Patent No. US Patent No. 6,858,430

Patent Title : An improved process for cultivation of algae

2

US Patent No 6,893,479 Indian Patent No.224,938 US Patent No. 7,208,189

: Integrated method for production of carrageenan and liquid fertilizer from fresh seaweeds : Low sodium salt of botanic origin

US Patent No. 7,067,568 Indian Patent Appl. No. 1280/DEL/2005 PCT Publ. No. WO 2011/027360 A1 Indian Patent Appl. No.1839/DEL/2009 US Patent No. 8,252,359

: Process of preparation of biodegradable films from semi refined kappa-carrageenan : A process for integral production of ethanol and seaweed sap from Kappaphycus alvarezii. : Method for the preparation of refreshing drink and use thereof

US Patent Pub No US 2005/0267296 A1 Indian Patent Appl. No. 1189/DEL/2004

Cost-effective process for preparing agarose from Gracilaria spp.

3 4 5 6 7

:

9

PCT Patent Pub. No.: WO2010109289 A8 Indian Patent Appl. No. IN2009DE567A PCT Pub. No. PCT WO2014027368 A3

10

Provisional Patent Appl No. 3194/DE/ 2012

11

PCT Pub. No. WO2014167583 (A1) : Kappaphycus alvarezii sap free of gibberellic acid (GA3) and its US Patent No. 2015/0232438 A1 method of preparation US Patent No. US 10,000,579 B2 June 2018 : An integrated process to recover a spectrum of bioproducts from Indian Patent Appl. No. 3811DEL2013 fresh seaweeds. Provisional Patent Appl. No. 1520DEL2015 : A novel method for the production of graphene sheets with tunable functionalities from seaweeds using deep eutectic solvents. A scalable process for liquid phase exfoliation of graphite to Provisional Patent Appl. No. 4344/DEL/ 2015

8

12 13

14

: An improved process for the extraction of agarose from seaweed extractive. : Process for improved seaweed biomass conversion for fuel intermediates and fertilizer Preparation of multifunctional hydrophobic ropes from kappa carrageenan

Dwindling harvest imperils global agar ICT-Mumbai

World harvest country wise 120

Food grade

Bacteriological grade 100

Production in MT

100

91

80

75

60 45

46

44

44

44

42

40

38

40 20

18

19

18

16

18

18

17

16

35

15

33

13

12

12

15

0

Agar production in India Source: M/s. Madurai Agar Industries Association, Tamilnadu

INDIA ICT-Mumbai

Demographic details of people involved in seaweed collection and seaweed industry in India Seaweed collection Place Palk bay

Mallipattinam

Valinokkam

Villages Female

Male

24

460

210

Gulf of Mannar

14

1270

285

Total

38

1730

495

Total annual value of seaweeds harvested from wild stocks Rs. 27 crores Agar industries

25

Alginate industries

7

Total

32

Annual Production: • Agar : 250-270 tons • Alginates: 320-340 tons

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Landings (Drytones)

600

Harvesting of seaweeds from natural beds for industrial processing Gelidiella acerosa Gracilaria edulis

500 400 300 200 100 0

Harvesting of Gelidiella acerosa Years

Landings (Drytones)

Landings of Agarophytes 3500 3000 2500 2000 1500 1000 500 0

Sargassum Turbinaria

Harvesting of Sargassum

Years

Landings of Alginophytes

Harvesting of Turbinaria

Status of Seaweed Farming in India ICT-Mumbai

Commercial Scale Tamil Nadu

Pilot Scale Gujarat

Kappaphycus seaweed technologies fuelling rural economy and agricultural productivity ICT-Mumbai

Commercial farming well established in Tamilnadu providing employment to about 2000 people especially women with an additional income of ₹ 15,000/- per month per person

₹ 1400 crores additional income to farmers

350 300

325

250

200

900

700

650

320

50

280

100

525

150

950

200

950

250

150

Income of farmers (Rupees in Lakhs)

400

0

Year

Employment and income generation from seaweed farming Product Impact [through Ms. AquAgri Processing Pvt. Ltd.] • 7.5 million liters of biostimulant manufactured. • The biostimulant has benefited over 3.5 million acres farming • Since 2015, biostimulant worth 300,000 $ exported

Extensive field applications: across 20 states – across crops. Yield increase of 11-36% over and above recommended dose of fertilizers (RDF) and recommended practices for the respective crops by liquid seaweed fertilizer

Sulfohydrolase from Gracilaria dura ICT-Mumbai Sulfohydrolase

Agarose

Agar

Fig. SEM image (A) Control (B) Sulfohydrolase treated agar Fig. FT-IR spectra of A. control agar and B. Sulfohydrolase treated agar

205 kDa

Table. Effect of additives on Sulfohydrolase activity

97 kDa

Additive

66 kDa

Control Cu Ca

Residual activity 100 78.71±3.8 121.3± 3.8

Mg

139.8± 3.7

Na

106.86± 4.7

K

100.58 ± 3.15

Hg Pb PMSF CHCl3 n-Hexane

ND ND ND 102± 3.58 90.67± 2.72

Toluene Cyclohexane

63.33±2.7 44 ± 3.77

EDTA

59.7 ± 3.14

43 kDa 29 kDa

Table. Comparison of native and enzyme treated agar Agar type Gel Gelling Melting strength temperature temperature (g cm-2) Commercial agar

190.0 ± 20.0

Enzyme treated agar

486.7 ± 15.3

(º C)

(º C)

39 ± 1.5

90.67 ± 3.1

Sulfate

Viscosity

(%)

(Cp)

ΔSulfate a (%)

3,6-AG content (%)

2.8 ± 0.2

9.7 ± 1.5

18 ± 0.9 60.59

31 ± 1.0

82.67 ± 2.5

Shukla et al. Carbohyd. Polym. 2011, 85:157-163

1.1 ± 0.1

18.0 ± 2.0

30 ± 0.98

a: Δ Sulfate (%): decrease in sulfate content

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BioPotash

MOP: 30-40% on dry weight basis of seaweed

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Cultivation of Porphyra (Nori) began before 260 years in Japan

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Processing of nori sheets shown in the traditional picture

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Pole system in Ariake-kai in Japan Net size, 2 m x 18 m

Harvesting by boat at floating cultivated system ICT-Mumbai

Porphyra nursery ICT-Mumbai

Monostroma nursery ICT-Mumbai

Source: Seaweed cultivation and marine ranching

Laminaria nursery ICT-Mumbai

Source: Seaweed cultivation and marine ranching; https://umaine.edu/cooperative=aquaculture

Mass production of micro-propagules from embryogenic pigmented callus of Kappaphycus ICT-Mumbai

1mm

5mm

250m

Explant with callus

3mm

Callus with micropropagules

Subcultured callus

Propagules of Kappaphycus

10cm

Kappaphycus habit

3cm

200m

Embryogenic callus embedded in agar plate

Filamentous embryogenic callus

Mass culture of propagules in flasks

US Patent No 6,893,479 Reddy et al., J. Phycol. 39, 610-616, 2003

3cm

Laboratory grown Field grown Kappaphycus plantlets of Kappaphycus 10cm

2cm

Young plantlets of Kappaphycus

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A model for large scale production of seedlings Gupta et al. Algal Res. 2018. 31:463-468

Seeding Tank Each side was seeded with the protoplasts isolated from 100 mg tissue/biomass

Reusability of enzyme

No. of Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle

Initial Biomass (mg) 100 100 100 100 100

Residual Biomass (mg) 1set 2 set 30 42 56 58 48 49 57 53 55 57

Propagation Tank Protoplasts Yield 1set 2.0 × 107 8.1 × 106 1.4 × 106 8.9 × 105 2.0 × 105

2 set 3.2 × 106 2.3 × 106 2.8 × 106 1.4 × 106 7.5 × 105

Regeneration Rate (%) 1set 2 set 91±32 92±54 89±15 87±23 89±50 88±45 90±45 84±23 81±23 80±45

Bioproces technology for integrated recovery of multiple products from marine macroalgal feedstock ICT-Mumbai

US Patent No. US 10,000,579 B2 2018 Indian Patent Appl. No. 3811DEL2013 Baghel et al. 2015. Green Chem. 17: 2436

Product yield and their market value at one ton fresh biomass

Quantity

Market value (₹)

Crude pigments (kg)

0.4-1.0

6,120-5,300

Distinct advantages of process

Plant nutrient rich sap (L)

300-600

18,000-36,000

   

Crude lipid (kg)

1.4-4.8

192-662

Agar (kg)

27-94

22,000-5,000

Cellulose (kg)

5-42

500-4200

Products

Recovery of five products Densification of product Improved quality of agar Efficient utilisation of feedstock

Total market value of products

46,000-1,30,000

Market value of produce (FW)

5000-30,000

Production cost of produce

3000-14000

Macroalgal biorefinery: Green seaweeds ICT-Mumbai

~14% Trivedi et al. 2016. Sci. Rep.6:30728 Tejal et al. 2017. Bioresource . Technol. 243: 867-873

Sap Liquid fertilizer

Biorefinery Process ~10%

Residue

Residue

Residue

PUFA-MUFA

Lipids ~1.5%

Cellulose Residue

Nanotechnology Biofuel

DBT-ICT CEB

Proteins ~11%

~20% Ulvan

Polymer technology

Dietary Proteins

Bioactive peptides

Tissue engineering

Food hydrocolloid

Proximate composition Carbohydrate Protein lipid Moisture Ash Carbon Hydrogen Nitrogen Sulfer Cellulose

Relative % on dry weight basis 43±4.5 14.4±2.2 1.83±0 19.86±1.3 16±2.7 25.64±1.6 5.75±2.4 3.13±0.88 5.52±0.45 15±2.3

Marine macroalgal biorefinery: alginophytes Sargassum spp. FW 1 Kg

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Sargassum granule wet(373 g)

Zero effluent process

Sap (541 ml)

* Bleaching with NaClO2 Bleached biomass dry (61.5 g)

Product I

Hydrolyzed with NaCO3

Alginic Acid (32 g) *

Residue Treated with 0.5 M NaOH

Product II Filtrate Acid precipitation

* Protein Concentrate (3.8 g)

Product III

mg/ g Salt 825.56 ± 6.54 11.56 ± 1.02 20.84 ± 0.88 31.42 ± 1.93 0.42 ± 0.04 1.88 ± 0.12 2.73 ± 0.09 0.47 ± 0.03 0.37 ± 0.05 0.38 ± 0.05 0.67 ± 0.03 25.36 ± 1.60 59.25 ± 3.48

* Liquid effluent

Residue

*

Minerals NaCl KCl MgCl2 CaCl2 Co Cu Fe Mn Ni Se Zn Protein Carbohydrate

Acid treatment 5% (V/V)

Neutralized with NaOH

Salt (115 g)

Cellulose (10 g)

Product IV

Product V

Environmental impact assessment ICT-Mumbai

Overview of Presentation

 International Scenario  National Status  Road map for making accelerated growth A) Technical  Efficient cultivation methods  Abiotic factors  Farm management  Post harvesting strategies B) Scientific  Domestication  Improved and resilient strains  Nursery development  Environmental impact  Application of advanced knowledge  Downstream Process  Scale up process  Value addition  Green process  Seaweed Policy  Seaweed development board

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Book Publications ICT-Mumbai

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Thank You CSIR, New Delhi DSIR, New Delhi DBT, New Delhi DST, New Delhi GSBTM, Gandhinagar GLPC, Gandhinagar

…….. in pursuit of scientific and technological excellence for revolutionising the blue economy in the country