Effect of substratum topography on algal turf colonization and productivity under different nutrient conditions by Manjinder Kaur A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August 6, 2016 Keywords: Algae, Substratum characteristics, Nutrient concentration, 3 D printing, Biomass density Copyright 2016 by Manjinder Kaur Approved by David Blersch, Chair, Assistant Professor of Biosystems Engineering Andres Carrano, Associate Professor of Industrial Engineering Sushil Adhikari, Alumni Associate Professor of Biosystems Engineering ABSTRACT Algae can act as a promising source for biofuel production, pollution recovery from natural waters and nutrient recovery from wastewaters. Typical algae cultivation involves algae in the suspended form, and separation methods including flocculation, filtration, and centrifugation contribute to high cultivation costs. Benthic algae, which grow attached to a growth substratum, is a good alternative to suspended algae for cultivation, as algal biomass can be harvested using mechanical scraping and vacuuming. This approach, called algae turf scrubbers (ATS), have been used for benthic algae cultivation at a large scale in outdoor algae cultivation for pollutant recovery from natural waters and wastewaters. There is little control, however, over the environmental conditions (temperature, light intensity, nutrients and pH) in outdoor ATS systems, and design of the reactor components, such as the growth substratum topography characteristics, can be key to determining the quantity and quality of the biomass produced. The characteristics of the substratum topography can be altered to control the colonization of algae, maximize algal biomass densities, and determine species selectivity to affect the quality and quantity of biomass. The objective of this research is to test the effect of substratum surface topography, using additive manufacturing (AM) technology to prototype, on the biomass density (biomass per unit area) and species selectivity under varying nutrient concentrations (low, medium and high). Substratum test samples were designed using hemispheres of 500µm, 1000µm and 2000µm radius with AM technology and a plain surface was kept as control. Replicates of each of surface ii topography were made using clay. Four Algal species (Oedogonium crassum, Sirogonium sticticum, Microspora floccose and Mougeotia scalaris) were seeded into a laminar flow lane reactor, and cultivated under different nutrient treatments (low, medium, and high). Repeated harvests of algal biomass were analyzed for biomass density, ash content, and species abundance, and correlations between these parameters, surface topography, and nutrient treatment were investigated. Results demonstrated that nutrient concentration has a primary effect on algal biomass density. The highest nutrient concentration had 186% more biomass density than the lowest concentration (control) and 136% more than the medium concentration. Substratum topography had a secondary effect on the biomass density, and different surface topographies had different biomass densities under each nutrient concentration. The surface topography with 2000 µm radius hemispheres has the highest average biomass density (1.06 ± 0.53 mg/cm2) followed by the surface with 500 µm radius hemispheres (0.92 ± 0.41 mg/cm2) for seven day harvest period. Biomass from the medium nutrient concentration had the highest ash content (17.16% ± 0.71%), whereas the highest nutrient concentration had lowest ash content percent (14.11% ± 0.32%). Nutrient concentration also has a primary effect on the abundance of algal species in the system. At the lowest concentration, Microspora floccose was in abundance (40.00% ± 1%), and at medium nutrient concentration Microspora floccose (45.68% ± 0.76%) and Mougeotia Scalaris (43.50% ± 0.84%) were in abundance. Oedogonium crassum (34.14% ± 1.25%) and Sirogonium scalaris (39.14% ± 1.19%) were most abundant at the highest nutrient concentrations. Substratum characteristics affect the species abundance only at the lowest nutrient concentrations, where Microspora floccose was the only species out of the four affected by substratum characteristics, where it was observed to be more abundant on 500 µm radius hemispheres and 2000 µm radius iii hemispheres. These results demonstrate the efficacy of using substratum design to control biomass characteristics and quantity in attached growth algae cultivation systems iv ACKNOWLEDGEMENTS I would like to express my deep gratitude to my major advisor Dr. David Blersch for giving me this opportunity and for all the support, patience and guidance during the course of my study. I would also like to thank Dr. Andres Carrano for all his support and guidance in research work and data analysis. I would also like to thank Dr. Sushil Adhikari for his time, guidance and valuable suggestions. A special thanks goes to John and Doc for designing the incubator to conduct this research work. I am grateful to my research group Kamran Kardel, Gabriel Proano, Joseph Ekong, and Ali Khoshkhoo for all their support during the research work. Most importantly, I would like to thank my parents, S. Maghar Singh Dhaliwal and Mrs. Parmjit Kaur for their unconditional love, guidance and support throughout my life, as it would not be possible without them. I would also like to thank my younger brother Ravinder for his love, and support in my life. I am very thankful to my friends, Anupriya, Ramandeep, Pamal, Geetika and Gurdeep for their support and encouragement. v TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS .......................................................................................................................... v LIST OF FIGURES ...................................................................................................................................... x LIST OF TABLES ..................................................................................................................................... xiii Chapter 1 Introduction ............................................................................................................................... 1 Economic importance of Algae ............................................................................. 1 Problems with Wastewater treatment -Algae Biofuel production scenario .......... 1 Research justification ............................................................................................ 3 Goals and Objectives of research .......................................................................... 4 Chapter 2: Review of Literature ............................................................................................................... 5 Algae ..................................................................................................................... 5 Composition of Algal Biomass ............................................................................. 6 Use of algae in different fields .............................................................................. 8 2.3.1 Algal Biofuels .................................................................................................... 8 2.3.2 Algae in water treatment and Nutrient recovery ............................................... 8 2.3.3 CO2 bio fixation ................................................................................................. 9 Algae Cultivation Systems .................................................................................. 10 2.4.1 Open Ponds or Raceway ponds ....................................................................... 11 2.4.2 Photo Bioreactors ............................................................................................ 12 vi 2.4.3 Algal Turf Scrubbers ....................................................................................... 14 Factors affecting algal colonization and characteristics ...................................... 16 2.5.1 Temperature ..................................................................................................... 16 2.5.2 Light................................................................................................................. 17 2.5.3 pH .................................................................................................................... 18 2.5.4 Nutrients .......................................................................................................... 19 2.5.5 Substratum characteristics ............................................................................... 20 Use of 3D (Three Dimensional) or additive manufacturing (AM) printing in the biological field ...................................................................................................... 24 2.6.1 Using 3D printing or AM technology to replicate surface roughness ............. 26 Current limitations in algae industry ................................................................... 29 Chapter 3: Material and Methods ........................................................................................................... 31 Introduction to the Study ..................................................................................... 31 Experimental Set Up ........................................................................................... 32 3.2.1 Preliminary experimentation on Flow lane incubator ..................................... 35 Three Dimensional printing of tiles their replication using clay ......................... 38 3.3.1 Set up of the tiles in the incubator ................................................................... 39 Algae collection and seedling the incubator ....................................................... 41 Nutrient concentrations selected for the experiment ........................................... 42 Initial start of the incubator and its daily operations ........................................... 44 Biomass harvesting, storage and restarting the incubator ................................... 44 vii 3.7.1 Restarting the incubator after each harvest ...................................................... 46 3.7.2 Restarting the incubator after each treatment .................................................. 47 Biomass Analysis ................................................................................................ 48 3.8.1 Biomass density ............................................................................................... 48 3.8.2 Percentage Ash Content ................................................................................... 50 Water Chemistry Analysis .................................................................................. 52 Microscopic Identification .................................................................................. 53 Simpsons Diversity Index ................................................................................... 55 Statistical analysis ............................................................................................... 55 Chapter 4: Results and Discussion ......................................................................................................... 57 Introduction ......................................................................................................... 57 Experimental results: Objective 1 ....................................................................... 58 4.2.1 Biomass Density .............................................................................................. 58 4.2.2 Percent ash content .......................................................................................... 63 Experimental Results: Objective 2 ...................................................................... 65 4.3.1 Microspora floccose ........................................................................................ 66 4.3.2 Mougeotia scalaris ........................................................................................... 67 4.3.3 Oedogonium crassum ...................................................................................... 69 Relative abundance of algal species at different surface topography in all three treatments………………………………………………………………………...72 4.4.1 Treatment 1 ...................................................................................................... 72 viii 4.4.2 Treatment 2 ...................................................................................................... 73 4.4.3 Treatment 3 ...................................................................................................... 75 Relative abundance of all four algal species at each surface topography in different nutrient treatments ................................................................................................ 76 4.5.1 Plain surface .................................................................................................... 76 4.5.2 Substratum with 500μm radius hemispheres ................................................... 78 4.5.3 Substratum with 1000 μm radius hemispheres ................................................ 79 4.5.4 Substratum with 2000 μm radius hemispheres ................................................ 81 Simpson’s Diversity index (SI) ........................................................................... 82 Chapter 5: Summary and Future Recommendations ........................................................................... 85 Summary and discussion ..................................................................................... 85 Conclusions ......................................................................................................... 90 Contribution of Research work and Future Recommendations .......................... 91 References ................................................................................................................................................. 93 ix LIST OF FIGURES Figure 2.1: Arial view of raceway pond (Chisti, 2007). .......................................................... 12 Figure 2.2: Schematic of a closed photo bioreactor system. ................................................... 13 Figure 2.3: Lab Scale ATS System (Adey and Loveland, 1998). ........................................... 15 Figure 2.4: ATS on the Great Wicomico River off the Central Chesapeake Bay (Adey et al, 2013). .................................................................................................................... 15 Figure 2.5: Gelatin-based micro-3D printing in the presence of bacteria (Connel et al, 2013). . ……………………………………………………………………………………26 Figure 2.6: Comparison of ceramic tiles and printed plastic tiles in natural streams (Kardel et al., 2015). .............................................................................................................. 27 Figure 2.7: Circular and rectangular tiles before and after biofilm colonization in an ATS. .. 28 Figure 2.8: Biomass density on different surfaces of both the tiles. ....................................... 28 Figure 3.1: Flow lane incubator and frame assembly. ............................................................. 33 Figure 3.2: Schematic diagram of flow lane incubator with dimensions. ............................... 34 Figure 3.3: Flow velocity at different intervals of each flow lane when set to 0.03 L s-1. ...... 36 Figure 3.4: Schematic diagram of heat map generation of flux values at each tile of four flow lanes. ..................................................................................................................... 37 Figure 3.5: 3D printed plastic tile (left) and its replicated clay tile with substratum having radius of hemispheres (a) 2000 µm, (b) 1000 µm and (c) 500 µm. ...................... 38 Figure 3.6: Pattern of different clay tiles in the flow lane photo incubator............................. 40 Figure 3.7: Placement of tiles on the flow lane photo incubator. ............................................ 41 Figure 3.8: Algal species used for seeding the incubator (a) Sirogonium sticticum, (b) Mougeotia scalaris, (c) Oedogonium crassum and (d) Microspora floccose. ...... 42 Figure 3.9: Different location to get algae for microscopic work. .......................................... 45 Figure 3.10: Storage of algal biomass in VWR formalin vial. .................................................. 45 Figure 3.11: Vacuum harvesting apparatus (left) and vacuuming process (right). ................... 46 x