Characterization of the use of wastewater from hydrothermal

liquefaction as a nitrogen source by the potential algal biofuel strain

Picochlorum sp.

Shuyi Wang, Xinguo Shi, Fatima Foflonker, Debashish Bhattacharya, Brian Palenik

Scripps Institution of Oceanography University of California, San Diego

Development of a sustainable nutrient recycling system

Needed: microalgae that grow on raffinate

What are raffinates?

3http://algae.illinois.edu/Projects/Hydrothermal.html

Gas

Organic phase“Oil”

Solid

Aqueous phase “Raffinates”

Hydrothermal liquefaction extractable liquid fraction

Screened strains for growth on raffinate including microalgal community in a local pond.

4(Wang, et al. J. Phycol., 2014)

Picocystis

Picochlorum

Chaetoceros

What is Picochlorum SENEW3?

• Isolated from San Elijo Lagoon pond

• Chlorophyta,Trebouxiophyceae• Green, oval, ~2uM diameter,

flagella and pyrenoid absent, autosporulation

• High growth rate, 0.5/day at 22 °C• Makes lipid droplets under

nitrogen stress• Able to use organic nitrogen

sources

5

Picochlorum SENEW3

Picochlorum grows in 10%-300% seawater

6

Picochlorum nitrogen recycling

7

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

50

100

150

200

250

300

11-May 16-May 21-May 26-May 31-May 5-Jun

RFU

or

mg

/ L

RFU

DW

OD

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

11-May 16-May 21-May 26-May 31-May 5-Jun

Cel

ls /

ml

Raffinate

Picochlorum (isolated from SElagoon)700 L seawater+100L inoculantAir bubbling and circulating800mL raffinate added at the beginning160mL raffinate added twice

Photo by Billy Lambert

provided by Billy Lambert

TN PO4

11936 3072 N:P=26:1

Add 160mL raffinate

Growth of Picochlorum in pond

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0

50

100

150

200

250

300

350

400

6-May 16-May 26-May 5-Jun 15-Jun 25-Jun 5-Jul 15-Jul 25-Jul

RFU

or

mg

/ L

Picochlorum SE3

RFU

DW

OD

June 242nd harvest

June 71st harvest

8

provided by Billy Lambert

TN mass balance calculation

• 1st harvest on June 7th

9

0.53g in water

Input: 13.44g

Wastewater (including cells): 5.45g

Algae clay: 5.36g

1.76g in water

Input: from recycling 5.45g + adding raffinate 3.84g = 9.29g

Wastewater (including cells): 7.67g

Algae clay: 2.70g

• 2nd harvest on June 24th

TN data provided by Nicole Heaps

Oil Content: 38%Recovery rate: 40~80%

Genome and Proteomics Data summary

Picochlorum SE3

• Genome size 13.5 Mbp* in collaboration with

• Debashish Bhattacharya (Rutgers Univ.)

• Protein number: 7367

iTRAQ proteomics data for 4 samples

• Protein identified (FDR 5%): 2836

• Protein identified (with more than one unique peptide identified at 95% confidence): 2418

10

*Reference: Foflonker et al. Environmental Microbiology 2014

Experimental setup

• Greenhouse bags

• Biological duplicates

• Exponential phase cells

• French pressed cells

• Raffinate composition (below)

With raffinate With nitrate (control)

Bag A Bag B Bag A Bag B

iTRAQ114

iTRAQ115

iTRAQ116

iTRAQ117

Trypsin digestion

LC-MS/MS

ProteinPilot 3.0

NH4-N, 6973, 58%

NO3-N, 16.7, 0%

NO2-N, 58.7, 1%

TKN, 4887, 41%

TN=11936 ppm

11

Data analysisMost strict Cut-off criteria:• All 4 ratios between replicates are significantly changed

(p<0.05)• More than 1 unique peptide at 95% confidence• Ratio >1.2 or <0.8Protein Identified: 56 (Set A)

For a less strict Cut-off criteria:• At least 1 ratio between replicates are significantly changed

(p<0.05)• More than 1 unique peptide at 95% confidence• Ratio >1.2 or <0.8Protein Identified: 383 (Set B)

12

---NA---protein(contig_41.g106.t1)carbamoyl-phosphate synthasedehydroascorbate reductaseornithine aminotransferasena+ k+ alpha polypeptidesuccinyl- ligasenucleoside-diphosphate-sugar epimerasepreprocathepsin caconitate mitochondrial-likemitochondrial nad-dependent malate dehydrogenasespore coat assembly proteinkynurenine 3-monooxygenase and related flavoprotein monooxygenaseshypothetical protein VOLCADRAFT_104245 mercaptopyruvate sulfurtransferase-like proteinlight-harvesting protein of photosystem il-ascorbate peroxidasecitrate synthasealanine-anticapsin ligaselight-harvesting chlorophyll a b-binding proteinchaperonin 60b2magnesium chelatase subunit of protochlorophyllide reductasecf1 alpha subunit of atp synthaseproteinatp synthase gamma-subunittcp-1 cpn60 chaperonin family protein40s ribosomal protein s8dcp1-like decapping family proteingeranylgeranyl reductaseglycosyltransferase-like proteintranslocation protein sec62protein(contig_139.g613.t1)protein(contig_45.g319.t1)60s ribosomal protein l1130s ribosomal protein s4nucleolar protein 1040s ribosomal protein s3ribosomal protein s20cf0 atp synthase subunit ii precursor60s ribosomal proteinprotein(contig_144.g757.t1)rna helicase60s ribosomal protein l9proliferation-associated protein 2g4-likesec14 cytosolic factor family protein phosphoglyceride transfer family proteinprotein(contig_56.g251.t1)4-aminobutyrate aminotransferaserossmann-fold nad -binding domain-containing proteinalanine aminotransferaseg-protein coupled inwardly rectifying k+ channelat dna binding proteinchitin deacetylase 5 isoform b precursorflagellar associated protein40s ribosomal protein s1550s ribosomal protein l34kynurenine 3-monooxygenase and related flavoprotein monooxygenasesef-hand domain-containing thioredoxinaldehyde-alcohol dehydrogenase glutamine synthetase

Color Key

-1 -0.5 0 0.5 1

Row Z-Score

16 17 14 15

Control Raffinate

Set A

Compared to Control

• 25 down regulated

• 31 up regulated

• Raffinate up-regulated proteases

Proteomic data: 1 up-regulated in Set A and 11 up-regulated in Set BConfirmed by AMC enzyme assay.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

FINDINGS

Pro

teas

e ac

tivi

ty (

nm

ole

AM

C/c

ell/

min

)

To utilize peptides in Raffinate?

FINDINGS

• Raffinate caused oxidative stress to cells

Proteomic data:

significant up-regulation of several antioxidant enzymes

(e.g. l-ascorbate peroxidase up 7.0; superoxide dismutase up 3.9)

15

Confirmed byAscorbate Peroxidase (AsPOX) assay

16

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Exponential Stationary

μm

ol a

sco

rbic

aci

dm

g p

rote

in1

min

1

Nitrate

HTL-WW

Due to toxic material in raffinates?

Conclusions

• Picochlorum grows robustly on “raffinates” as a nitrogen source, including in outdoor ponds.

• “Omics” techniques show that Picochlorumchanges protein expression growing on raffinate.

• It experiences oxidative stress when growing on raffinate; this knowledge may help screen for additional species with the ability to grow on raffinate or engineer such strains.

Acknowledgements

18

• Shuyi Wang

• William Lambert

• Sophia Giang

• Emy Daniels

• Xinguo Shi

• Stilianos Roussis

• Richard Cranford

• Nicole Heaps

• Nathan Schoepp

• Dominick MendolaRUTGERS University

•Debashish Bhattacharya•Fatima Foflonker