Thesis for the Degree of Doctor of Philosophy Integration of filamentous fungi in ethanol dry-mill biorefinery Jorge A. Ferreira Copyright © Jorge A. Ferreira, 2015 Swedish Centre for Resource Recovery University of Borås SE-501 90 Borås, Sweden Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-674 ISBN 978-91-87525-77-3 (printed) ISBN 978-91-87525-78-0 (pdf) ISSN 0280-381X Skrifter från Högskolan i Borås, nr. 73 Cover: Dark field microscopy photograph of submerged growth of Neurospora intermedia Printed in Sweden Repro-service: Responstryck AB Borås, 2015 ii ABSTRACT The industrial production of bioethanol as a replacement to gasoline is well-established worldwide, using starch- or sugar-rich substrates. Additionally, the bioethanol plants produce animal feeds derived from fermentation leftovers. The biorefinery character of bioethanol plants can be enhanced via process diversification. This entails the production of more value- added products, which can be accomplished by including edible filamentous fungi as the second biocatalysts while taking advantage of the available equipment for cost-effective inclusion. The process diversification can be achieved either via valorisation of the process leftovers or via inclusion of other residual substrates. In dry-mill biorefineries, baker’s yeast is unable to consume residual pentose sugars and other more complex substrates in the process leftovers so called whole stillage and thin stillage. Edible ascomycetes and zygomycetes fungi can be used to accomplish yeast and consume those residual substrates in stillage as well as from external substrates of lignocellulosic origin, e.g. spent sulphite liquor and wheat straw. The conversion of these substrates to ethanol, and biomass rich in protein, lipids, respective essential amino acids and fatty acids as well as chitosan was investigated in this thesis. Among the filamentous fungi studied, Neurospora intermedia was the best ethanol producer from thin stillage. Process developments included primary shake-flasks experiments, followed by pilot scale-up using 26 L, 2.3 m3 and 80 m3 bioreactors. The 26 L bioreactor, as a bubble column led to similar performance as an airlift bioreactor, and also a continuous mode could be successfully used instead of a batch process. By using a dilution rate of 0.1 h-1, around 5 g/L of ethanol and 4 g/L of biomass rich in protein, lipids, amino acids and fatty acids essential to humans were obtained. The inclusion of the process can potentially lead to a spent medium lower in solids and viscosity which may facilitate the energy-intensive evaporation and drying steps as well as the water recycling back to the process. By applying a two-stage cultivation with whole stillage, up to 7.6 g/L of ethanol could be produced using 1 FPU cellulase/g suspended solids and 5.8 g/L of biomass containing 42% (w/w) crude protein. In the first stage (ethanol production), N. intermedia was used, while Aspergillus oryzae was the biocatalyst in the second stage for further biomass production. Both strains were able to degrade complex substrates both in liquid and solid fraction of whole stillage. The extrinsic substrates included spent sulphite liquor and pretreated wheat straw slurry. When the former was used, up to around 7 g/L of Rhizopus sp. could be obtained in a 26 L airlift bioreactor. The biomass was rich in protein and lipids (30–50% and 2–7% on a dry weight basis, respectively). The monomers of the latter were continuously filtered for production of biomass under simultaneous saccharification, fermentation and filtration. Biomass yields of up to 0.34 g/g of consumed monomeric sugars and acetic acid were obtained. The inclusion of the process for valorisation of thin stillage can potentially lead to the production of 11,000 m3 ethanol and 6,300 tonnes of biomass at a typical facility producing 200,000 m3 ethanol/year. Keywords: airlift bioreactors, ascomycetes, biomass, bubble column, ethanol, feed, Neurospora intermedia, thin stillage, zygomycetes iii iv LIST OF PUBLICATIONS This thesis is mainly based on the results presented in the following articles: I. Ferreira, J. A., Lennartsson, P. R., Niklasson, C., Lundin, M., Edebo, L., Taherzadeh, M. J., Spent sulphite liquor for cultivation of an edible Rhizopus sp. Bioresources. 2012: 7, pp. 173-188. II. Ferreira, J. A., Lennartsson, P. R., Edebo, L., Taherzadeh, M. J., Zygomycetes- based biorefinery: Present status and future prospects. Bioresource Technology. 2013: 135, pp. 523-532. III. Ferreira, J. A., Lennartsson, P. R., Taherzadeh, M. J., Production of ethanol and biomass from thin stillage using food-grade Zygomycetes and Ascomycetes filamentous fungi. Energies. 2014: 7, pp. 3872-3885. IV. Ferreira, J. A., Lennartsson, P. R., Taherzadeh, M. J., A pilot study on production of ethanol and biomass from thin stillage by Neurospora intermedia for process diversification. Engineering in Life Sciences. DOI: 10.1002/elsc.201400213. V. Bátori, V., Ferreira, J. A., Taherzadeh, M. J., Lennartsson, P. R., Ethanol and protein from ethanol plant by-products using edible fungi Neurospora intermedia and Aspergillus oryzae. Submitted. VI. FazeliNejad, S., Ferreira, J. A., Brandberg, T., Lennartsson, P. R., Taherzadeh, M. J., Fungal protein and ethanol from lignocelluloses by Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Manuscript. v STATEMENT OF CONTRIBUTION Jorge A. Ferreira’s contributions to each of the above publications are: Paper I: Responsible for most of the experimental work, data analysis and writing of the manuscript. Paper II: Responsible for most of the literature survey, data collection and most of the writing of the manuscript. Paper III: Responsible for part of the idea, most of the experimental work, data analysis and most of the writing of the manuscript. Paper IV: Responsible for part of the idea, most of the experimental work, data analysis and most of the writing of the manuscript. Paper V: Responsible for part of the idea, part of the experimental work, data analysis and most of the writing of the manuscript. Paper VI: Responsible for part of the experimental work, data analysis and part of the writing of the manuscript. vi NOMENCLATURE AFEX Ammonia fibre expansion AIM Alkali-insoluble material ALB Airlift bioreactor BC Bubble column CDS Condensed distiller’s solubles CSTR Continuous-stirred tank reactor DDGS Distiller’s dried grains with solubles DHA Docosahexaenoic acid DM Dry matter EPA Eicosapentaenoic acid GHG Greenhouse gases GlcN Glucosamine GlcNAc N-acetyl-glucosamine GRAS Generally regarded as safe GYV Glucose, yeast extract and vitamin LCAs Life cycle assessments PM10 Particulate matter s.d. Standard deviation SHF Separate hydrolysis and fermentation SS Suspended solids SSF Simultaneous saccharification and fermentation SSFF Simultaneous saccharification, fermentation and filtration SSL Spent sulphite liquor SSL50% Spent sulphite liquor diluted to 50% Vvm Volume of air per volume of medium per minute WO Wet oxidation vii viii TABLE OF CONTENTS 1 INTRODUCTION .............................................................................................................. 1 1.1 Preface and scope ........................................................................................................ 1 1.2 Thesis outline ............................................................................................................... 3 1.3 Oil refineries ................................................................................................................ 3 1.4 Biorefineries ................................................................................................................ 6 1.5 Ethical and Social Aspects .......................................................................................... 8 2 1st Generation bioethanol Biorefineries ............................................................................ 11 2.1 (Bio)Ethanol .............................................................................................................. 11 2.2 1st generation bioethanol production plants ............................................................... 12 2.3 Process diversification ............................................................................................... 14 3 Potential Substrates for process diversification of 1st generation bioethanol plants ......... 17 3.1 Substrates intrinsic to the process .............................................................................. 17 3.1.1 Whole stillage and thin stillage .......................................................................... 18 3.2 Substrates extrinsic to the process ............................................................................. 20 3.2.1 Wheat straw ........................................................................................................ 21 3.2.2 Spent sulphite liquor (SSL) ................................................................................ 23 4 Filamentous Fungi as Potential bioCatalysts for biorefineries ......................................... 29 4.1 The kingdom Fungi ................................................................................................... 29 4.2 Baker’s yeast .............................................................................................................. 31 4.3 Filamentous fungi ...................................................................................................... 31 4.3.1 An overview