Hochschule Rhein-Waal Rhine-Waal University of Applied Sciences Faculty of Life Sciences Production and Chromatographic Analysis of Polyhydroxybutyrate from waste product streams by Azohydromonas lata A Thesis Submitted in Partial Fulfilment of the Requirements of the Degree of Bachelor of Science in Bioengineering By Dominic Kösters Matriculation Number: 19056 Supervisors: Prof. Dr. Joachim Fensterle Prof. Dr. Peter Simon Submission Date: 04.11.2019 i Executive Summary As conventional plastic production receives much criticism for being harmful to both humans and the environment, this work focused on establishing a batch fermentation based on waste substrates from local industries to produce polyhydroxybutyrate (PHB) with Azohydromonas lata (A. lata). Additionally, the produced PHB was characterised by qualitative and quantitative gas chromatography coupled to a mass spectrometer (GC- MS) and gel permeation chromatography (GPC). Multiple substrates, including rhubarb waste, pea waste, and dairy industry wastewater (KAAS medium), were tested for successful fermentation. Rhubarb and pea waste could not be used as a fermentation substrate due to obstacles in upstream processing. KAAS medium was prepared by centrifugation and sterile filtration and tested in a 1 L and 4 L fermentation yielding non- competitive biomass and PHB concentrations with 0.09 g/L and 0.004 g/L, respectively. A 3 L fermentation with pure saccharose as the carbon source served as a model for comparison and analysis and resulted in a measured biomass concentration of 3.58 g/L with a PHB content of 1.43 g/L after Soxhlet extraction. The GPC analysis led to no conclusive elograms and results due to issues in non-destructively extracting and solving the PHB. The GC-MS analysis showed excellent linearity with an R2 value of 0.9906 for the standard curve and clear chromatograms, which allowed for both qualitative and quantitative analysis of PHB. Since PHB concentrations from KAAS medium were low, no quantitative analysis could be performed. Only samples from the saccharose-based fermentation could be assessed quantitatively. The found maximum PHB concentration was 2.95 g/L, which deviated from the found 1.43 g/L after Soxhlet extraction and was therefore discussed in this work. No locally available waste substrate could be found to be suitable for an industrial scale fermentation. The findings during this work led to the conclusion that semi-sterile fermentation approaches shall be investigated in future works handling waste products as they can potentially overcome upstream processing steps and, hence, make rhubarb or pea waste a potential candidate for PHB production. ii Acknowledgement I am grateful for the assistance given by my supervisors Prof. Dr. Joachim Fensterle and Prof. Dr. Peter Simon, who supported and motivated me during my thesis. I am particularly thankful to Ralf Lucassen and Dr. Stefan Weber for their constant constructive support and the time they invested in this work. Additionally, I would like to thank Chika Igwe and Felicia Zlati for providing their knowledge on the production of biopolymers from microorganisms. iii Table of Contents Contents 1 Introduction ................................................................................................................ 1 1.1 Current Situation ................................................................................................. 1 1.2 Biopolymers ........................................................................................................ 2 1.2.1 Poly-3-hydroxybutyrate (PHB) Classification ................................................. 3 1.3 PHB Application .................................................................................................. 4 1.4 PHB Production in Azohydromonas lata .............................................................. 5 1.5 PHB Analysis ...................................................................................................... 7 1.6 Substrates ........................................................................................................... 8 1.7 Objective ............................................................................................................. 9 2 Materials and Methods ............................................................................................. 10 2.1 Chemicals and Media ........................................................................................ 10 2.2 Equipment ......................................................................................................... 12 2.2.1 Devices and Apparatus ............................................................................... 12 2.2.2 Software ..................................................................................................... 13 2.3 Data Assessment .............................................................................................. 13 2.3.1 Optical Density (OD) ................................................................................... 13 2.3.2 Dry Cell Weight ........................................................................................... 13 2.3.3 Biomass Concentration ............................................................................... 14 2.3.4 Total Cell Counts ........................................................................................ 14 2.3.5 Growth Parameters ..................................................................................... 14 2.3.6 pH ............................................................................................................... 15 2.3.7 Dissolved Oxygen (DO) .............................................................................. 15 2.3.8 Antifoam Control ......................................................................................... 15 2.4 Bacterial Strain .................................................................................................. 16 2.5 Substrates and Preparation ............................................................................... 16 2.5.1 Dairy Industry Wastewater .......................................................................... 16 2.5.2 Rhubarb Waste ........................................................................................... 16 iv 2.5.3 Pea Waste .................................................................................................. 16 2.5.4 Saccharose Medium ................................................................................... 16 2.6 Substrate Analysis ............................................................................................. 16 2.7 Fermentation ..................................................................................................... 17 2.7.1 Pre-Culture ................................................................................................. 17 2.7.2 Main-Culture Flask Fermentation ................................................................ 17 2.7.3 Main-Culture Bioreactor Fermentation ........................................................ 17 2.8 Polymer Extraction and Purification ................................................................... 18 2.8.1 Polymer Extraction 3 L and 7 L Scale-Up Fermentation .............................. 18 2.9 Polymer Analytics .............................................................................................. 18 2.9.1 Fluorescence Microscopy ........................................................................... 18 2.9.2 Spectrofluorometry...................................................................................... 18 2.9.3 Gel Permeation Chromatography (GPC) ..................................................... 18 2.9.4 Gas Chromatography-Mass Spectrometry (GC-MS) ................................... 19 3 Results .................................................................................................................... 21 3.1 1 L KAAS Medium Fermentation ....................................................................... 21 3.1.1 Sugar Composition ..................................................................................... 21 3.1.2 Supplementation for Optimal Growth Conditions ......................................... 21 3.1.3 Growth Assessment .................................................................................... 23 3.2 3 L Saccharose Fermentation ........................................................................... 25 3.2.1 Growth Assessment .................................................................................... 25 3.2.2 Spectrofluorometric Analysis ....................................................................... 27 3.2.3 PHB Extraction ........................................................................................... 28 3.3 7 L KAAS Fermentation ..................................................................................... 29 3.4 GC-MS Analysis of Fermentation Products ....................................................... 30 3.4.1 Qualitative PHB Analysis ............................................................................ 30 3.4.2 Quantitative PHB Analysis .......................................................................... 32 3.4.3 Analysis of KAAS Medium Samples ............................................................ 35 3.5 GPC Analysis of Fermentation Products ........................................................... 36 3.6 Rhubarb Waste Fermentation ..........................................................................