Characterization of the use of wastewater from hydrothermal liquefaction as a nitrogen source by the potential algal biofuel strain Picochlorum sp.

Transcription

1 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

2 Development of a sustainable nutrient recycling system Needed: microalgae that grow on raffinate

3 What are raffinates? Gas Solid Hydrothermal liquefaction Organic phase Oil extractable liquid fraction Aqueous phase Raffinates 3

4 Screened strains for growth on raffinate including microalgal community in a local pond. Picochlorum Picocystis Chaetoceros (Wang, et al. J. Phycol., 2014) 4

5 What is Picochlorum SENEW3? 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

6 Picochlorum grows in 10%-300% seawater 6

7 Cells / ml RFU or mg / L Picochlorum nitrogen recycling Add 160mL raffinate RFU DW OD Raffinate TN Photo by Billy Lambert PO N:P=26: May 1.00E May 21-May 26-May 31-May 5-Jun 9.00E E E+07 Picochlorum (isolated from SElagoon) 700 L seawater+100l inoculant Air bubbling and circulating 800mL raffinate added at the beginning 160mL raffinate added twice 6.00E E E E E E+07 provided by Billy Lambert 0.00E May 16-May 21-May 26-May 31-May 5-Jun 7

8 RFU or mg / L Growth of Picochlorum in pond 400 Picochlorum SE RFU DW OD June 7 1 st harvest June 24 2nd harvest May 16-May 26-May 5-Jun 15-Jun 25-Jun 5-Jul 15-Jul 25-Jul provided by Billy Lambert 8

9 TN mass balance calculation 1 st harvest on June 7 th Input: 13.44g Oil Content: 38% Recovery rate: 40~80% Algae clay: 5.36g 0.53g in water 2 nd harvest on June 24 th Wastewater (including cells): 5.45g Input: from recycling 5.45g + adding raffinate 3.84g = 9.29g Algae clay: 2.70g 1.76g in water Wastewater (including cells): 7.67g TN data provided by Nicole Heaps 9

10 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 *Reference: Foflonker et al. Environmental Microbiology

11 Experimental setup With raffinate With nitrate (control) Bag A Bag B Bag A Bag B itraq 114 itraq 115 itraq 116 itraq 117 Greenhouse bags Biological duplicates Exponential phase cells French pressed cells Raffinate composition (below) TN=11936 ppm Trypsin digestion LC-MS/MS ProteinPilot 3.0 NO2-N, 58.7, 1% TKN, 4887, 41% NO3-N, 16.7, 0% NH4-N, 6973, 58% 11

12 Data analysis Most 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 <0.8 Protein 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 <0.8 Protein Identified: 383 (Set B) 12

13 Color Key Control Raffinate Row Z-Score Set A Compared to Control 25 down regulated 31 up regulated NA--- protein(contig_41.g106.t1) carbamoyl-phosphate synthase dehydroascorbate reductase ornithine aminotransferase na+ k+ alpha polypeptide succinyl- ligase nucleoside-diphosphate-sugar epimerase preprocathepsin c aconitate mitochondrial-like mitochondrial nad-dependent malate dehydrogenase spore coat assembly protein kynurenine 3-monooxygenase and related flavoprotein monooxygenases hypothetical protein VOLCADRAFT_ mercaptopyruvate sulfurtransferase-like protein light-harvesting protein of photosystem i l-ascorbate peroxidase citrate synthase alanine-anticapsin ligase light-harvesting chlorophyll a b-binding protein chaperonin 60b2 magnesium chelatase subunit of protochlorophyllide reductase cf1 alpha subunit of atp synthase protein atp synthase gamma-subunit tcp-1 cpn60 chaperonin family protein 40s ribosomal protein s8 dcp1-like decapping family protein geranylgeranyl reductase glycosyltransferase-like protein translocation protein sec62 protein(contig_139.g613.t1) protein(contig_45.g319.t1) 60s ribosomal protein l11 30s ribosomal protein s4 nucleolar protein 10 40s ribosomal protein s3 ribosomal protein s20 cf0 atp synthase subunit ii precursor 60s ribosomal protein protein(contig_144.g757.t1) rna helicase 60s ribosomal protein l9 proliferation-associated protein 2g4-like sec14 cytosolic factor family protein phosphoglyceride transfer family protein protein(contig_56.g251.t1) 4-aminobutyrate aminotransferase rossmann-fold nad -binding domain-containing protein alanine aminotransferase g-protein coupled inwardly rectifying k+ channel at dna binding protein chitin deacetylase 5 isoform b precursor flagellar associated protein 40s ribosomal protein s15 50s ribosomal protein l34 kynurenine 3-monooxygenase and related flavoprotein monooxygenases ef-hand domain-containing thioredoxin aldehyde-alcohol dehydrogenase glutamine synthetase

14 Protease activity (nmole AMC/cell/min) FINDINGS Raffinate up-regulated proteases Proteomic data: 1 up-regulated in Set A and 11 upregulated in Set B Confirmed by AMC enzyme assay. To utilize peptides in Raffinate?

15 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

16 μmol ascorbic acid mg protein1 min1 Confirmed by Ascorbate Peroxidase (AsPOX) assay Nitrate HTL-WW Exponential Stationary Due to toxic material in raffinates? 16

17 Conclusions Picochlorum grows robustly on raffinates as a nitrogen source, including in outdoor ponds. Omics techniques show that Picochlorum changes 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.

18 Acknowledgements Shuyi Wang William Lambert Sophia Giang Emy Daniels Xinguo Shi RUTGERS University Stilianos Roussis Richard Cranford Nicole Heaps Nathan Schoepp Dominick Mendola Debashish Bhattacharya Fatima Foflonker 18