DESIGN ASPECTS OF SOLID STATE FERMENTATION A thesis submitted to the UNIVERSITY OF MANCHESTER for the degree of DOCTOR OF PHILOSOPHY in the Faculty of Engineering and Physical Sciences by MUSAALBAKRI ABDUL MANAN Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science Faculty of Engineering and Physical Sciences University of Manchester 2014 The Microbe The microbe is so very small You cannot make him out at all But many sanguine people hope To see him through a microscope His jointed tongue that lies beneath A hundred curious rows of teeth His seven tufted tails with lots of lovely pink and purple spots On each of which a pattern stands Composed of forty separate bands His eyebrows of a tender green All these have never yet been seen But Scientist who ought to know Assure us that they must be so Oh, let us never, never doubt What nobody is sure about Hilaire Belloc 1870 – 1953 DESIGN ASPECTS OF SOLID STATE FERMENTATION ABSTRACT Solid state fermentation (SSF) refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment to which the selected microorganisms, especially fungi, are naturally adapted. The current status of SSF research globally was discussed in terms of articles publication. This was followed by discussion of the advantages of SSF and the reason for interest in SSF as a notable bioprocessing technology to be investigated and compared to submerged fermentation (SmF) for the production of various added-value products. SSF also proved to be a potential technology to treat solid waste produced from food and agricultural industry and to provide environmental benefits with solid waste treatment. A summary was made of the attempts at using modern SSF technology for future biorefineries for the production of chemicals. Many works were carried out in the Satake Centre for Grain Process Engineering (SCGPE), University of Manchester, to prove the strategy of using SSF for the production of hydrolysate rich in nutrients for sequel microbial fermentation with or without adding any commercial nutrients. The research findings presented in this thesis are based on a series of SSF experiments carried out on systems varying in complexity from simple petri dishes to our own design of bioreactor systems. They were conducted to assess a solution for biomass estimation, enzymes production, and successful mass and heat transfer. A proper technique for inoculum transfer prior to the start of the fermentation process was developed. In SSF, estimation of biomass presents difficulties as generally the fungal mycelium penetrates deep and remains attached with the solid substrate particles. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become laborious, impractical and inaccurate. Essentially, this remains another critical issue for monitoring growth. In these studies, measurement of colour changes during SSF are presented as one of the potential techniques that can be used to describe growth, complementary to monitoring metabolic activity measurement, such as CER, OUR and heat evolution, which is directly related to growth. For the growth of Aspergillus awamori and Aspergillus oryzae on wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. This colourimetric technique was also proved to be a feasible approach for fungal biomass estimation in SmF. This new approach is an important complementation to the existing techniques especially for basic studies. The key finding is that the colourimetric technique demonstrated and provided information of higher quality than that obtained by visual observation or spores counting. The effect of aeration arrangements on moisture content, oxygen (O ), mass and heat transfer during SSF was investigated. 2 A. awamori and A. oryzae were cultivated on wheat bran in newly designed four tray solid state bioreactor (SSB) systems. The new tray SSB systems were: (1) single circular tray SSB, (2) multi-stacked circular tray SSB, (3) Single rectangular tray SSB and (4) multi-square tray SSB. The purpose was to study the effect, on heat and water transfer, of operating variables, fermentation on the perforated base tray and internal moist air circulation under natural and forced aeration. Temperature, O and carbon dioxide were measured continuously on-line. Enzyme activity, moisture content and biomass 2 were also measured. The results suggest that the air arrangements examined have a remarkable effect on the quantity of biomass produced using measurement of spores and enzymes production. The strategy presented in these studies allowed quantitative evaluation of the effect of forced internal moist air circulation on the removal of metabolic heat. With the proposed strategy, it was possible to maintain the bed temperatures at the optimum level for growth. However, the effect on moisture content was very different for the two fungi. It was found that the ability of A. oryzae to retain moisture was much higher than that of A. awamori. This is possibly due to the higher levels of chitin in A. oryzae. Greater spores and enzymes (glucoamylase, xylanase and cellulase) production was observed for A. awamori in multi-stacked circular tray and multi-square tray SSB systems compared to the conventional petri dishes and the other two systems. A. oryzae was excellent in producing protease in the same bioreactors. A direct technique of establishing a correlation between fungal growth and CER, OUR, heat evolved was proven successful in this work. The information obtained from CER and OUR led to the estimation of respiratory quotient (RQ). RQ describes the state of the fungal population in the tray SSB and gives an indication of fungal metabolic behaviour. RQ values < 1 were obtained from 38 experiments using four tray SSB systems for the two fungi. A kinetic model based on CO evolution 2 instead of biomass concentration was examined in order to simplify the required experiments for kinetic model development. A Gompertz model was used to fit the integrated CO data and predict the quantity of CO evolution in all 2 2 experiments. A correlation was found between the heat evolution and CER. The performances of tray SSB systems can be improved by constructing them as multi-trays. The multi-tray systems improved the mass transfer considerably compared with single tray systems. In addition, the multi-tray systems allowed precise measurement of the gradients of CO , 2 enzymes, spores and fungal biomass. In addition, the air arrangements using moistened air were successful in maintaining moisture content, adequate O supply and control of temperature, and hence, increased the productivity of both fungi. 2 Overall A. awamori and A. oryzae have their own ability and performance to degrade and utilise the complex compositions contained in the solid substrate and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray and multi-square tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large scale operation, though considerable optimisation work remains to be done, for example the height/diameter ratio and total number of trays should be optimised. MUSAALBAKRI ABDUL MANAN PhD THESIS 2014 ii TABLE OF CONTENTS PAGE ABSTRACT i DECLARATION viii COPYRIGHT STATEMENT viii ACKNOWLEDGEMENT ix CHAPTER 1: INTRODUCTION 1 1.1 BACKGROUND 1 1.2 PROBLEM STATEMENT 2 1.3 THESIS STRUCTURE 3 CHAPTER 2: SOLID STATE FERMENTATION: A REVIEW OF THE 7 LITERATURE 2.1 INTRODUCTION 7 2.2 DEFINITION OF SSF 9 2.3 SSF – CURRENT STATE AND PERSPECTIVES 10 2.4 PERCEIVED ADVANTAGES OF SSF 17 2.5 DISADVANTAGES OF SSF 17 2.6 THE DIFFERENCE BETWEEN SSF AND SmF 19 2.7 FACTORS THAT INFLUENCE SSF 20 2.7.1 Biological Factors 21 2.7.1.1 The type of microorganism 21 2.7.1.2 Inoculum 22 2.7.1.3 Substrates 22 2.7.2 Physico-chemical Factors 24 2.7.2.1 Moisture content 24 2.7.2.2 pH 25 2.7.2.3 Temperature 26 2.7.2.4 Gaseous environment 26 2.7.2.5 Aeration 26 2.7.2.6 Particle size 27 2.7.3 Mechanical Factors 27 2.7.3.1 Agitation/Mixing 27 2.7.3.2 Particular design of bioreactors 28 2.8 ESTIMATION OF GROWTH IN SSF 28 2.8.1 Measuring Cell Components not Present in the Substrate 29 2.8.2 Measuring Biomass Components Present in Both Substrate and Biomass 29 2.8.3 Measuring Other Secondary Metabolites 30 2.8.4 Measuring Metabolic Activity 30 2.8.5 Measuring Images through Direct Microscopic Observation 31 2.8.6 Measuring Biomass from the Solid Matrix 32 2.8.7 Other Techniques 32 2.9 BIOREACTOR FOR SSF 35 2.9.1 Classification of Bioreactors for SSF 36 2.9.1.1 Group 1 41 2.9.1.2 Group 2 42 iii 2.9.1.3 Group 3 43 2.9.1.4 Group 4 44 2.10 MASS TRANSFER PHENOMENA IN SSF 55 2.10.1 Micro-scale Phenomena 55 2.10.1.1 Inter-particle mass transfer 57 2.10.1.2 Intra-particle mass transfer 59 2.10.1.3 Heat transfer 59 2.10.1.4 Water transfer 60 2.10.2 Macro-scale Phenomena 61 2.11 SSF BIOPROCESSING – BASED BIOREFINERY DEVELOPMENT 62 2.12 CONCLUDING REMARKS 67 CHAPTER 3: PROJECT OBJECTIVES AND PLANS 69 CHAPTER 4: EXPERIMENTAL DESIGN AND RESEARCH PLAN 75 4.1 INOCULUM TRANSFER PROCEDURES 75 4.2 PETRI DISH EXPERIMENTS 79 4.3 BIOMASS MONITORING 79 4.3.1 Organic Matter Loss 79 4.3.2 Dry Weight Reduction Ration 80 4.3.3 Biomass Monitor 80 4.3.4 Metabolic Measurements 82 4.3.4.1 Oxygen uptake rate and carbon dioxide evolution rate 82 4.3.4.2 Heat evolution 83 4.3.5 Colourimetric Technique 84 4.4 TRAY STUDIES 84 4.5 TRAY SOLID STATE BIOREACTOR STUDIES 85 4.5.1 Single Circular Tray Solid State Bioreactor 86 4.5.2 Multi-stacked Circular Tray Solid State Bioreactor 89 4.5.3 Single Rectangular Tray Solid State Bioreactor 91 4.5.4 Multi-square Tray Solid State Bioreactor 93 4.5.5 Bioreactor Set Up 96 CHAPTER 5: RESEARCH AND ANALYTICAL TECHNIQUES 99 5.1 MICROORGANISM 99 5.2 PREPARATION OF STANDARD INOCULUMS 100 5.2.1 Monospore Isolation Technique 100 5.2.2 Fungal Inoculums Preparation 100 5.3 SUBSTRATE 102 5.4 MOISTURE CONTENT 103 5.5 SPORE COUNTS 103 5.6 SAMPLE EXTRACTION 103 5.7 GLUCOSAMINE 104 5.8 TOTAL REDUCING SUGARS 105 5.9 GLUCOSE 107 5.10 FREE AMINO NITROGEN 108 5.11 pH 110 5.12 ENZYMES 110 5.12.1 Glucoamylase Activity 110 5.12.2 Protease Activity 111 iv 5.12.3 Xylanase Activity 112 5.12.4 Cellulase Activity 114 CHAPTER 6: STUDIES OF PHYSICAL CHARACTERISATION OF SOLID 119 STATE FERMENTATION 6.1 INTRODUCTION 119 6.2 MATERIALS AND METHODS 120 6.2.1 Evaluating Particle Size 120 6.2.2 Properties of Solid Substrate 122 6.2.3 Water Retention Value 124 6.2.4 Aeration Studies 126 6.3 RESULTS AND DISCUSSION 128 6.3.1 Evaluating Particle Size 128 6.3.2 Properties of Solid Substrate 130 6.3.3 Water Retention Value 134 6.3.4 Aeration Studies 141 6.4 SUMMARY AND CONCLUSIONS 147 CHAPTER 7: ESTIMATING GROWTH IN SOLID STATE 149 FERMENTATION 7.1 INTRODUCTION 149 7.2 MATERIALS AND METHODS 150 7.2.1 Microorganism, Inoculum and SSF 150 7.2.2 Biomass Measurement 150 7.2.2.1 Organic matter loss, dry weight reduction ratio and biomass 150 monitor 7.2.2.2 Metabolic measurements 150 7.2.2.3 Colourimetric technique 150 7.3 RESULTS AND DISCUSSION 152 7.3.1 Organic Matter Loss 152 7.3.2 Dry Weight Reduction Ratio 153 7.3.3 Biomass Monitor 156 7.3.3.1 Capacitance measurement using biomass monitor in SmF 156 7.3.3.2 Capacitance for biomass measurement in SSF 158 7.3.4 Metabolic Measurements 159 7.3.5 Colourimetric Technique 163 7.3.5.1 Preliminary petri dish experiments 165 7.3.5.2 Effect of extraction techniques on the quality of colour from 169 A. awamori 7.3.5.2.1 Effect of initial moisture content 169 7.3.5.2.2 Effect of inoculum size 172 7.4 SUMMARY AND CONCLUSIONS 175 CHAPTER 8: COLOUR DEVELOPMENT AS A POTENTIAL TECHNIQUE 177 FOR ESTIMATING FUNGAL GROWTH 8.1 INTRODUCTION 177 8.2 MATERIALS AND METHODS 178 8.2.1 Microorganisms and Inoculum Preparation 178 v 8.2.2 Fermentation Medium and Media Preparation 178 8.2.2.1 Solid agar medium 178 8.2.2.2 Solid state fermentation 178 8.2.2.3 Submerged fermentation 179 8.3 ANALYTICAL METHODS 180 8.3.1 Colour Extraction 180 8.3.1.1 Samples from solid agar media 180 8.3.1.2 Samples from SSF 180 8.3.1.3 Samples from SmF 181 8.3.2 Spectrophotometric Analysis 181 8.3.3 Biomass Estimation 182 8.4 RESULTS AND DISCUSSION 183 8.4.1 Effect of Different Solid Agar Media on Growth, Sporulation and Colour 184 Production of A. awamori and A. oryzae 8.4.2 Correlation between Spores Concentration and Colour Density 188 8.4.3 Correlation between Organic Matter Loss and Colour Density 190 8.4.4 Correlation between Glucosamine Concentration and Colour Density 192 8.4.5 Correlation between Enzyme Activities and Colour Density from SSF 195 8.4.6 Colour Development in SmF using Solid Substrates (I) 196 8.4.7 Colour Development in SmF using a Synthetic Medium (II) 199 8.5 SUMMARY AND CONCLUSIONS 202 CHAPTER 9: ENZYMES PRODUCTION STUDIES IN SINGLE TRAY 205 SYSTEM 9.1 INTRODUCTION 205 9.2 MATERIALS AND METHODS 206 9.2.1 Microorganisms, Preparation of the Inoculum and the Substrates 206 9.2.2 The Tray System 207 9.2.3 Moisture Content and Spores Count 207 9.2.4 Preparation of Enzymes Supernatant 207 9.3 RESULTS AND DISCUSSION 208 9.3.1 Moisture Content 208 9.3.2 pH Profile in SSF 209 9.3.3 Biomass Estimation 210 9.3.4 Total Reducing Sugars and Free Amino Nitrogen 211 9.3.5 Enzymes Production in SSF using in the Tray System 214 9.3.5.1 Glucoamylase 214 9.3.5.2 Protease 216 9.3.5.3 Xylanase 217 9.3.5.4 Cellulase 220 9.3.6 Mass Transfer Phenomenon in the Tray System 222 9.4 SUMMARY AND CONCLUSIONS 225 CHAPTER 10: BIOREACTOR STUDIES 227 10.1 INTRODUCTION 227 10.2 MATERIALS AND METHODS 228 10.2.1 Microorganisms, Inoculum and Substrate Preparation 228 10.2.2 The Tray Solid State Bioreactor 228 10.2.2.1 Initial moisture content arrangement 229 10.2.2.2 Air arrangement 229 10.3 RESULTS AND DISCUSSION 233 vi