1. Microalgae for valorization of industrial wastes from pulp/paper mills and oil refineries Algae Biomass Summit 2014 A. Kuehnle, N.A. Nolasco, M. Perez, M. Johnson

2. 2 O VERVIEW : W OOD W ASTE FROM P ULP /P APER M ILLS Bleached Southern Pine hydrolysate Southern Hardwood Chips hydrolysate Under USDA BRDI grant, Domtar teamed with KAS to valorize underutilized wood waste using microbial conversion into bioproducts. Domtar Largest uncoated freesheet (UFS) and paper grade pulp manufacturer in North America Recently added diversity to its product mix, ventured into consumer products Goal: “Fuel to feed” Hog fuel into feed (and higher value algal bioproducts) Wood hydrolysates produced by CSI, Cellulose Sciences International

3. P ROCESS Screen and select strains from KAS collection for growth on mixed sugars, tolerance to inhibitors in wood hydrolysates Establish algal biomass productivity and sugar utilization (HPLC) using aerobic fermentation Nutrient medium targeted biomass productivity (feed value and co-products) Batch fermentation conditions T= 30°C, pH= 7.0, agitation = 300 rpm, DO 2 = 100%, air = 3.0 L/min Hawaiian species, including Chlorella and Scenedesmus 3

4. 4 Hydrolysates profile: Salinity ranged 3.3% – 4.2%, pH: 5.5; undefined components Screening and controls: Standardized with C5 and C6 sugar equivalents W OOD HYDROLYSATES FROM CSI

5. G ROWTH V ARIED WITH E ACH T YPE OF W OOD HYDROLYSATE All five wood hydrolysates supported growth of algae 5

6. W OOD H YDROLYSATES O UTPERFORM M ODEL S UGARS A 1.6-fold higher biomass productivity (2.87 g/L-day), was obtained with bleached southern pine (BSP) hydrolysate than with C5 and C6 model sugars alone (1.7 g/L-day). 6

7. O THER O UTCOMES 7 C5 sugar effect Some strains thrive in hydrolysate vs in equivalent glucose alone Excellent sugar utilization, varies with hydrolysate and impurities 2:1 sugars : biomass ratio (SHC), EtOH present as impurity Beneficial non-sugar components present in CSI hydrolysates SHC BSP Specific sugar utilization of algae in two wood hydrolysates 2:1 5:1

8. C ONCLUDING T HOUGHTS Opportunity to develop mill-based strategies matching hog fuel with strains Can select preferred strain with lowest sugar requirement Select strain that benefits from non-sugar components present Select strains to utilize both the C5 and C6 sugars Co-cultivate strains to match wood composition, efficiency Product options for biomass Align volumes with off-take potential Preferably match mill products with regional processors and off-takers Next steps Validate metrics at next scale to meet targets in financial models 8

9. Algae-wastewater pilot facility was designed, constructed and operated in 2013-2014 at Chevron Hawaii to demonstrate nitrogen and phosphorous reduction in refinery wastewater Reduction in total suspended solids (TSS) in the effluent was also addressed, using tangential flow filtration Utilized waste CO 2 from refinery’s hydrogen formation plant, as previously demonstrated by KAS in EPA-award winning project 9 O VERVIEW : W ASTEWATER FROM O IL R EFINERIES

10. A LGAE P ILOT P LANT PBR AND S IMULATION T ANK PBR used Clear-Flex polyurethane tubing with sterile fittings fabricated to spec for KAS by Siftex (CT) Simulation tank (10-feet deep) used basal pneumatic mixing with plant air and CO 2 on discontinuous basis System designed for future deployment into existing basins for minimal footprint 10

11. E FFLUENT AND T EST P ROTOCOL Algae Pilot Initial TestAmerica: NH3-N, NO3-NO2 N, TKN, Total N, Total P KAS: NH3-N, NO3-N, PO4-P, algal count, bacterial CFU, DW (or TSS), DO, pH, temp During the Test (KAS) NH3-N, NO3-N, PO4-P algal count, bacterial CFU, DW (or TSS), DO, pH, temp Final TestAmerica: NH3-N, NO3-NO2 N, TKN, Total N, Total P KAS: NH3-N, NO3-N, PO4-P, algal count, bacterial CFU, DW (or TSS), DO, pH, temp 11 Chlorella-like Scenedesmus sp. Bacteria Triplicate grab samples- before and after treatment- were analyzed by TestAmerica

12. Wastewater rich in ammonia N N and P levels significantly reduced in 3 d 97% reduction in Ammonia N, 69% reduction in Total N, 90% reduction in Total P 12 Total N NH3 N Total P 69% Reduction: 97%90%

13. Scenedesmus thrived in the wastewater, compatible with the other microbes Previous work by KAS with this and other tested organisms showed utilization of mixed N sources, and variable amounts of NH 3 and NO 3 Very important for fluctuating oil refinery wastewater profiles Met end objective of using mixed microbes to best polish the wastewater 13 KAS740

14. Hollow fiber filtration removed 99+% of total suspended solids; suited to solids recycle as needed Basal pneumatic mixing system effective for deep ponds, promising for use in existing basins For managing nutrient loads and suspended solids: Results support final polishing of wastewater using KAS’s basis of design and P&ID solution Used off-the-shelf industrial equipment and components in innovative manner for ease of deployment 14 O THER O UTCOMES

15. Using KAS solution, possibility to address targets in both downstream and upstream pits/basins of the O&G industry Potential $10 B O&G wastewater market in U.S. by 2025 Additional validation, deployment with engineering firm Difficult to sell into this market without a partner 15 C ONCLUDING T HOUGHTS