ABSTRACT IMPROVED MICROALGAL BIOMASS HARVESTING USING OPTIMIZED ENVIRONMENTAL CONDITIONS AND BACTERIAL BIOFLOCCULANTS DEREK CONTE MANHEIM The cost and energy balance of microalgae biofuel production is sensitive to the algae harvesting method, among many other factors. Bioflocculation and settling of suspended microalgae cultures is a harvesting method with potentially low cost and energy input. However, bioflocculation (the spontaneous flocculation of algal cells without chemical addition) has not been a reliable process with cultures grown in ponds. To provide insights to help improve algae settling, factors affecting the settling of algae were investigated in the laboratory using pure cultures of two common microalgae species: Scenedesmus sp. and Chlorella vulgaris. Bioflocculation of these algae was studied with and without the addition of bioflocculants produced by the bacterium, Burkholderia cepacia, to improve settling efficiencies. The bioflocculant produced by this bacterium was used in two different forms: a cell suspension including capsular and dissolved extracellular polymeric substance (EPS) components of B. cepacia, and dialyzed filtrate of the bacterial culture (only dissolved EPS fraction). The effects of algal growth phase, mixing time, bioflocculant dose, and environmental conditions such as pH and nutrient deprivation of bacterial bioflocculant cultures on settling of the algae species were studied. Settling characteristics were different for the two algae cultures, and their settling was affected differently by the many factors studied. Scenedesmus settling was best in later growth stages, while Chlorella settled much better in early growth phases. Addition of B. cepacia cells as a bioflocculant improved settling of Scenedesmus, with the greatest effect during mid to late exponential growth of the Scenedesmus. In contrast, addition of B. cepacia filtrate as a bioflocculant best improved Chlorella settling during stationary growth of Chlorella. Longer mixing times (contact time between the algae cells and bacterial bioflocculant) improved the settling of Scenedesmus, while Chlorella settled better with a shorter mixing time. Reducing the pH to 3 (a typical isoelectric point for microalgae) improved the settling of both algae cultures, with and without bioflocculant addition. Increasing the pH to 11 autoflocculated Scenedesmus cultures, but not Chlorella cultures, at early growth stages. EPS produced by the algae, bacteria, and wastewater organisms was quantified using dialysis separation followed by total organic carbon (TOC) analysis. Wastewater organisms were included because wastewater is a potential growth medium for biofuel algae. Improved settling of both species of algae depended on both the quantity and type of EPS (dissolved or capsular) produced by both the bacterial bioflocculant, and the algae themselves. Scenedesmus settled the best during late growth phases while its own EPS production was high, and combined EPS (capsular and dissolved) from B. cepacia improved settling at a higher dosage of bacterial cells to algae (1:2 B. cepacia cells to algae cells). Since Chlorella settling was not improved at later growth stages when its own EPS production was greatest, it appears that Chlorella’s settling rate was less affected by the production of its own EPS. For Chlorella, B. cepacia EPS addition (capsular and dissolved) was effective only in low doses (1:6 B. cepacia cells to algae cells). Settling results with the addition of bacterial bioflocculants with the pure algae cultures were compared to settling results of lab experiments with algae pondwater sampled from high-rate algae ponds (HRAPs). These algae samples were used to test the addition of return activated sludge (RAS) to improve settling. RAS addition improved the settling of Chlorella, which was the dominant algae species in the HRAP during the time of this study, at two different doses (a ratio of RAS to algae pond water of 1:3 and 1:6). Nutrient deprivation of B. cepacia cells before use as a bioflucculant was found to improve settling for Scenedesmus, especially during early phases of growth when EPS production of Scenedesmus was low. The EPS produced by the starved bacterial cells was about 30% greater than that produced by cultures which were not nutrient-limited. For the bacterial cultures, EPS production peaked at mid stationary phase for non-starved cultures and during early stationary phase for starved cultures. Chlorella settling improved in early growth with starved bacterial cell addition and in later growth with non-starved bacterial cell addition. These results suggest that the settling of microalgae can vary dramatically by species and that the settling of different species is affected differently by growth phase and environmental conditions. In addition, species of algae respond differently to addition of bacterial bioflocculants. Given the dramatically different settling behavior of the two species of algae used in this research, more research should be directed to studying settling of other microalgal species. Based on this research, the use of bacterial bioflocculants is promising for improving algae settling and may contribute to the development of a reliable, low cost harvesting process for commercial biofuel production from microalgae.

Abstract The chemicals currently used for wastewater treatment are mainly based on synthetic inorganic or organic compounds. Oil-derived polyelectrolytes are used for the removal of colloidal solids from wastewater by flocculation and coagulation, for example, while activated carbon adsorbents are typically used to remove soluble impurities such as heavy metals and recalcitrance organic matter. Many of these chemicals have associated negative health impacts, and use of activated carbon has proved to be expensive. Moreover, the present synthetic chemicals are not readily biodegradable or renewable. Thus there is a high demand for “green” water chemicals which could offer a sustainable solution for achieving high-performance, cheap water purification. Water chemicals of a new type based on nano-scale particles (nanofibrils) derived from cellulose, i.e. nanocelluloses, are examined as possible bio-based chemicals for wastewater treatment. Two anionic nanocelluloses (dicarboxylic acid, DCC, and sulphonated ADAC) were tested as flocculants in the coagulation-flocculation treatment of municipal wastewater, while the flocculation performance of cationic nanocellulose (CDAC) was studied with model kaolin clay suspensions, and nanocelluloses produced from sulphonated wheat straw pulp fines (WADAC) were tested for the adsorption of lead (Pb(II)). The anionic nanocelluloses (DCC and ADAC) showed good performance in treating municipal wastewater in a combined coagulation-flocculation process with a ferric coagulant. In the case of both anionic nanocelluloses the combined treatment resulted in a lower residual turbidity and COD in a settled suspension with highly reduced total chemical consumption relative to coagulation with ferric sulphite alone. Likewise, the CDACs resulted in powerful aggregation of kaolin colloids and maintained effective flocculation performance over wide pH and temperature ranges. The capacity of the nanofibrillated and sulphonated fines cellulosics (WADAC) for the adsorption of Pb(II) was 1.2 mmol/g at pH 5, which is comparable to the capacities of commercial adsorbents. / Tiivistelmä Jätevesien kemiallinen käsittely pohjautuu pääsääntöisesti synteettisten epäorgaanisten ja orgaanisten kemikaalien käyttöön. Öljypohjaisia polyelektrolyytteja käytetään kolloidisten partikkeleiden poistamiseen jätevesistä koaguloimalla ja flokkuloimalla, kun taas liuenneita epäpuhtauksia, kuten raskasmetalleja, poistetaan useimmiten adsorboimalla ne aktiivihiileen. Synteettiset vesikemikaalit valmistetaan uusiutumattomista luonnonvaroista ja niiden hajoaminen luonnossa voi olla hidasta, minkä lisäksi monet näistä käytetyistä synteettisistä vesikemikaaleista ovat terveydelle haitallisia. Aktiivihiilen käyttö puolestaan on kallista, johtuen sen korkeista valmistus- ja käyttökustannuksista. Uusille ”vihreille vesikemikaaleille, jotka tarjoavat ympäristöystävällisempiä, halpoja sekä tehokkaita ratkaisuja vedenpudistukseen, onkin suuri kysyntä. Tässä työssä selluloosasta valmistettuja nanokokoisia partikkeleita, eli nanoselluloosia, on tutkittu yhtenä varteenotettavana biovaihtoehtona uusiksi kemikaaleiksi jätevesien puhdistukseen. Kahden anionisen nanoselluloosan (dikarboksyyli, DCC, ja sulfonoitu, ADAC) flokkauskykyä testattiin koagulointi-flokkulointi reaktioissa kunnallisen jäteveden puhdistuksessa. Kationisen nanosellun (CDAC) flokkauskykyä tutkittiin puolestaan kaoliinisaven malliliuoksilla ja vehnän korsisellun hienoaineista nanofibrilloimalla sekä sulfonoimalla valmistetuilla (WADAC) nanoselluloosamateriaaleilla testattiin lyijyn (Pb(II)) adsorptiota vesiliuoksista. Anioniset nanoselluloosat (DCC ja ADAC) toimivat tehokkaasti kunnallisen jäteveden flokkauksessa ferri-sulfaatin kanssa yhdistetyissä koagulointiflokkulointi reaktioissa. Yhdistetyissä reaktioissa molemmat anioniset nanoselluloosat vähensivät sameutta sekä COD pitoisuutta laskeutetuissa jätevesinäytteissä huomattavasti pienemmillä kemikaalikulutuksilla paremmin kuin pelkästään ferri-sulfaatilla koaguloitaessa. Myös CDAC:t toimivat tehokkaasti flokkauksessa keräten tehokkaasti kaoliinin kolloidipartikkeleita yhteen laajalla pH- ja lämpötila-alueella. Nanofibrilloidun ja sulfonoidun vehnäsellun hienoaineen (WADAC) adsorptiokapasiteetti lyijylle Pb(II) oli 1.2 mmol/g pH:ssa 5, mikä on verrannollinen kaupallisten adsorptiomateriaalien kapasiteettiin.