Co-Cultures of Yeasts and Zygomycetes in the Form of Pellets Methods for the Preparation of Pellets and Biocapsules, Their Properties and Applications

Co-cultures of Yeasts and Zygomycetes in the Form of Pellets Methods for the Preparation of Pellets and Biocapsules, Their Properties and Applications

Jonas Nyman Michael Lacintra

This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science with a Major in Recovery of Energy and Material Resources with specialisation in Industrial Biotechnology, 120 ECTS credits No. 1/2012 Co-cultures of Yeasts and Zygomycetes in the Form of Pellets

Jonas Nyman, [email protected] Michael Lacintra, [email protected]

Master thesis

Subject Category: Technology

University of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640

Examiner: Prof. Mohammad J. Taherzadeh

Supervisor,name: Dr. Patrik R. Lennartsson

Supervisor,address: University of Borås School of Engineering SE-501 90 BORÅS

Date: 2012-06-22

Keywords: Zygomycetes, Saccharomyces cerevisiae, Immobilization, Flocculation, Fungal Pellets, Biocapsules, Rhizomucor, Rhizopus, Mucor, Bioethanol, Fermentation

ii Contents

Abstract v

1 Introduction 1

2 Background 3 2.1 Pelleted morphology of ﬁlamentous fungi ...... 3 2.2 Factors believed to inﬂuence pelletization ...... 6 2.3 Co-cultures of fungi and yeasts ...... 12 2.4 Biocapsules ...... 12 2.5 Flocculation in yeasts ...... 13

3 Materials and Methods 15 3.1 Microorganisms ...... 15 3.2 Induction of pelleted growth ...... 15 3.3 Biocapsules ...... 16 3.4 Microscopy techniques ...... 16 3.5 Multi-factorial experiment ...... 21 3.6 Logistic regression ...... 22 3.7 Bioreactor fermentations ...... 22

4 Results 25 4.1 Formation of pellets ...... 25 4.2 Response surface optimization results ...... 26 4.3 Logistic regression results ...... 30 4.4 Substance 243 ...... 31 4.5 Biocapsules ...... 32 4.6 Results of Microscopy ...... 34 4.7 Results from bioreactor experiments ...... 35

5 Discussion 46 5.1 Pelletization ...... 46 5.2 Multi-factorial experiment ...... 46 5.3 Logistic regression ...... 47 5.4 Biocapsules ...... 47 5.5 Microscopy ...... 48

6 Conclusions 50

7 Suggestions for future work 50

8 Acknowledgements 51

References 52

Appendix A: Used media and chemicals 58

Appendix B: Central Composite Design 63

iii List of Figures

2.1 Interaction between yeast cell and mold hypae ...... 13 3.1 Equatorial image of pellet ...... 17 3.2 Biocapsules embedded in agar ...... 19 3.3 Microtome preparations of biocapsules ...... 20 3.4 Parafﬁn and agar sections ...... 20 4.1 Hairy pellet of M. indicus ...... 25 4.2 A pellet formed in a medium with CaCO3...... 26 4.3 Pellets formed in the factorial experiment ...... 27 4.4 Residual plot for response surface ...... 29 4.5 Residual plot for restricted model ...... 30 4.6 Biocapsules of Rhizomucor and red yeast ...... 33 4.7 Red yeast embedded in Rhizomucor biomass ...... 33 4.8 Rhizopus sp. stained with lactophenol cotton blue ...... 34 4.9 The inside of a pellet embedded in agar ...... 35 4.10 High resolution image of a microtome section of a pellet ...... 36 4.11 A biocapsule ...... 37 4.12 Yeast immobilized in fungal mycelium ...... 37 4.13 Yeast cells trapped in mycelium ...... 38 4.14 Flocs of yeast immobilized in a biocapsule ...... 38 4.15 Immobilized yeast in a microtome section...... 39 4.16 Focus stacked image ...... 39 4.17 Ethanol evaporation ...... 40 4.18 Results from fermentation with pellets of RM3 ...... 40 4.19 Results from fermentation with pellets of RM4 ...... 41 4.20 Bioreactors with limonene ...... 41 4.21 Long lag time with 0.25 % limonene ...... 42 4.22 Agar plate with yeast and Rhizomucor ...... 43 4.23 Fermentation of dilute acid hydrolysate ...... 44 4.24 Inhibitor degradation by Rhizomucor ...... 44 4.25 Biomass attached to bafﬂes ...... 45

iv Abstract

Many industrially important fungi can grow in the form of small spherical pellets. Such pellets reduce the viscosity and enhances mass transfer rates in culture broths. The pelleted morphology also influences the fungus’s metabolism, directing it to specific metabolites. The pellets are easily harvested from the broth and recycled. These properties makes pelleted morphology very attractive. The pelleted morphology of four Zygomycetes strains was studies. Several different nutrient media used by other researchers for achieving pelleted growth was tested. The influence of eight factors on pelletization of Rhizopus sp. in a completely defined medium was determined using a fractional central composite design and logistic regression. Pelleted growth of all four Zygomycetes was achieved, with very good results for two Rhizomucor sp. strains. A simple medium containing calcium carbonate was found to induce pelletization with very high reproducibility. Immobilization of yeast cells was attempted in pellets of Rhizomucor. It was found that a flocculating yeast can be immobilized inside pellets of fungal mycelium, forming ”biocapsules”. This is accomplished by first using a medium that induces pelletization of the filamentous fungus and does not allow for growth of the yeast. The pellets are then inoculated into a second medium that induces growth and flocculation of the yeast and inhibits further growth of the filamentous fungus. Non-flocculating yeasts could not be immobilized, suggesting that the flocculin proteins in the cell wall of flocculating strains are important for proper immobilization. The flocculation and immobilization arises due to expression of several different FLO-genes and the importance of these genes for successful immobilization is discussed. The results demonstrate that the morphology of Zygomycetes can be controlled and that this may be useful in industrial fermentation. The new immobilization technique reveals the great importance of flocculation and cell surface hydrophobicity. Using yeast strains that express certain FLO-genes may be beneficial in fermentation of lignocellulosic hydrolysates. Microscopy techniques were developed that allows for high quality microphotogra- phy of pellets and thin cross-sections of pellets and biocapsules.

v 1 Introduction

There are many good reasons for wanting to immobilize biomass in bio-reactors. Immobilization allows for higher dilution rates in continous fermentations, higher productivity and easier recycling of biomass. Many different techniques have been developed to achieve immobilization of cells (Verbelen et al. 2006). This thesis discusses a new method for immobilization of yeast cells inside spherical pellets of fungal biomass. Such pellets are potentially of great value in biotechnological processes, mainly because the pellet morphology greatly reduces the viscosity of the medium, which enhances mass transfer rates. This can lead to reduced costs for aeration, stirring and cooling in industrial processes (Gibbs et al. 2000, p. 19–20). Filamentous fungi can grow in several distinct morphological forms, from dispersed mycelia to irregular clumps and spherical pellets. Many industrially important species have been shown to sometimes agglomerate into pellets and grow in this form. The pelletization phenomenon is extremely complex and there is no coherent theory that explains it. Pellets have been studied for a long time and many factors affecting fungal morphology and pelletization are known (Papagianni 2004, Metz & Kossen 1977). It has been shown that addition of polymers and calcium ions to a medium can have a dramatic effect on the morphology and pellet formation, but the fundamental reasons for why this is the case remains unknown. The studies done on pelletization so far has been fairly simple and mainly qualitative. No previous studies have in a consistent manner studied interaction effects between the many factors influencing pelletization and most experiments have only tested one factor at a time. Both Ascomycota and Zygomycota can form pellets. In the literature they are often treated equally and the same factors are often assumed to affect both orders in the same way. Co-cultures of filamentous fungi and yeasts have received little attention, except in the case of rice wine fermentation and traditional fermented Asian foods. Yeasts, lactic acid bacteria and Zygomycetes can grow together, and are used to ferment tempeh, a traditional Indonesian food made by fermenting soy beans (Feng 2006, p. 41). In these fermentations enzymes excreted by the filamentous fungi hydrolyze the complex substrate into simple sugars that then become available to the yeast. These traditional mixed cultures are stable over long time and resistant to infections (Hesseltine et al. 1985). Pelleted co-cultures of co-immobilised Penicillium chrysogenum and Saccharomyces cerevisiae have been described and studied earlier (Peinado et al. 2006, Martínez et al. 2011, García-Martínez et al. 2012). It is well known that there is a very limited mass transfer into the pellets (Schügerl et al. 1983). This makes the pellets anaerobic in the center and larger pellets become hollow as the cells eventually undergo autolysis. Because of the very limited diffusion of nutrients and especially oxygen into the pellets, pelleted morphology has often been considered a disadvantage (Driouch et al. 2012, p. 467). However, the limited mass transfer might be beneficial if the medium is toxic. The reduced

1 diffusion might protect the cells from inhibitors. Lignocellulosic hydrolysates contain several inhibitory compounds that makes them difficult to ferment efficiently (van Maris et al. 2006). Mucor indicus have proven relatively resistant to inhibitors such as acetic acid, furfural, 5-hydroxymethylfurfural and limonene (Lennartsson et al. 2009). Zygomycetes are also good ethanol producers and their biomass might be used as fish feed, a high value product (Lennartsson et al. 2011). The possibility of immobilizing yeasts inside pellets of Zygomycetes is therefore attractive for several different reasons. In this thesis the pelletization of four different Zygomycetes, all of the order Muco- rales, is described. The possibility of encapsulating yeast cells inside the pellets was examined and the tolerance of such biocapsules for limonene was evaluated in batch fermentations.

2 2 Background

2.1 Pelleted morphology of ﬁlamentous fungi

2.1.1 Advantages of pellets

The rheological properties of fermentation media are important. Viscous and non- newtonian liquids suffer from poor mixing and mass transfer, and considerable eco- nomic savings can be achieved if the viscosity can be kept low during fermentation (Schügerl et al. 1983). Growth of filamentous fungi in the form of pellets does reduce the viscosity when compared to freely suspended mycelium. Driouch et al. (2012, p. 465) reported a four-fold reduction in viscosity, using Aspergillus niger. van Suijdam et al. (1980) also writes that the most important advantage is the reduced viscosity but also adds the greatly simplified separation of the biomass from the broth. There is considerable evidence for the fact that the morphology also affect the metabolism of the fungi, so that pelleted growth directs the metabolism to specific metabolites. For instance, the morphology of Rhizopus nigricans affects its ability to hydroxylate progesterone (Žnidaršicˇ et al. 1998). The influence of pelleted growth on the metabolism of Aspergillus terreus was studied by Bizukojc & Ledakowicz (2010). It has also been reported that pelleted growth is desirable for the production of itaconic acid, citric acid, penicillin (Driouch et al. 2012), lovastin (Casas López et al. 2005) and lactic acid (Yu et al. 2007).

2.1.2 Coagulation theory of pelletization

Takahashi & Yamada (1959) described two different kinds of fungal behavior and they suggested the classification of fungi in two classes, coagulative and non-coagulative fungi, depending on weather the spores agglomerated or not. Since then, others have tried to classify different species accordingly. For instance, A. niger is a coagulative fungi and P. chrysogenum is non-coagulative (Papagianni 2004, p. 201). However, the classification is ambiguous and more recent findings on the importance of media composition has revealed that pelletization is much more complex. Further- more, it seems there is no relationship between phylogeny and coagulation. Pellets are believed to form in either of five different ways: • In the so-called coagulative fungi the inoculated spores agglomerate/coagulate and then each agglomerate grow to form one pellet. • In non-coagulative fungi the spores do not coagulate, but germinate individu- ally. The young hyphae may then entangle and form small clumps that grow to form pellets. • If the inoculum concentration is small enough, the average distance between spores may be so large that each single spore grow to form one pellet. This is an extreme case of non-coagulative behaviour.

3 • The presence of polymers, calcium ions or other substances acts as a glue making spores or hyphae agglomerate. • Solid particles of calcium carbonate, titanate or other minerals acts as nucleation sites.

2.1.3 Kinetics of pelleted growth

Because of the limited diffusion rate in the pellets, growth occurs only in a thin layer on the surface of the pellet. The critical penetration depth is the depth of the biomass layer that is metabolically active. In Aspergillus niger and A. awamori it has been reported to be 185–205 µm (Driouch et al. 2012, 468). Cronenberg et al. (1994) directly measured the oxygen and glucose concentrations inside pellets of P. chrysogenum and found that in young pellets the oxygen concentration drops rapidly in a 70 µm outer layer. In older pellets, with a lower metabolic rate and higher diffusivity, the pellets were fully aerated. Glucose consumption in pellets cause a very low sugar concentration inside pellets regardless of age. There is a closely related critical pellet diameter. When the growing pellet reaches this diameter, the cells in the pellet’s center die because of nutrient starvation. Pellets larger than the critical pellet diameter therefore becomes hollow. It must be noted that pellets do not follow the usual Monod-kinetics. The biomass in a pellet grows according to equation 2.1.

p3 p3 M(t) = M0 + kc · t (2.1)

Where M(t) is the biomass at time t, M0 is the biomass at t = 0, denoting the time of the beginning of the growth phase and kc is a growth rate constant. The radius of a pellet increases linearly as equation 2.2.

r(t) = r0 + kr · t (2.2) Žnidaršicˇ et al. (1998, p. 211) determined the growth rate constants for R. nigricans ◦ 1/3 −1/3 −1/3 in an ordinary medium at 23 C to be kc = 0.037—0.048 g ·l ·h , where the lower value is for 100 rpm and the higher for 225 rpm. Radial growth rates were reported to be between 0.081 and 0.168 mm/h at 100 and 225 rpm respectively. The radial growth rate can be expressed as kr = wµ, where w is the thickness of the growing zone and µ is the specific growth rate. If there are many pellets in a culture, the larger specific surface area will result in a faster growth of biomass. To account for the number of pellets in a culture, equation 2.3 may be used. r4πρn M = M + 3 · wµt (2.3) 0 3 Where ρ is the pellet’s density and n the number of pellets. The study of pellets have suffered from the subjective and qualitative classification of pellet density, smoothness, ”hairyness” and other characteristics. However, there are methods for more objective measurements and determination of pellet morphology using computer analysis of images of pellets (Cox & Thomas 1992, Cox et al. 1998).

4 2.1.4 Fermentation of lignocellulosic hydrolysates

Lignocellulosic biomass is abundant in nature and it is the only resource that, on a large scale, can replace petroleum as a source of fuels and bulk chemicals. Much work has been done on trying to convert lignocellulose into convenient fuels, such as methanol, ethanol and butanol. The primary goal has been to efficiently produce ethanol in fermentative processes, using the lignocellulose as carbon source for some microorganism. Fermenting lignocellulosic materials is inherently difficult because of a few main problems. • Lignocellulose consists of three different polymers: cellulose, hemicellulose and lignin. The hydrolysis of any one of these occurs at conditions sub-optimal for the hydrolysis of the others. • The lignocellulose is physically hard to break. Cellulose molecules are intertwined into crystalline fibrils and fibers. These fibers sits in a highly branched and crosslinked matrix of hemicellulose and lignin. • The polymers are chemically resistant to hydrolysis. The β-glucanoside bonds in cellulose and hemicellulose have a sterical configuration which makes them resistant to enzymatic hydrolysis. • If the lignocellulose is hydrolyzed using dilute acids, toxic substances are formed, necessitating a detoxification before fermentation. • The hydrolysates contain several different sugars, including a substantial frac- tion of pentoses. There are very few microorganisms that can utilize the pentoses and produce a valuable product. Pichia stipitis is one notable yeast that can ferment xylose to ethanol. Baker’s yeast is a very efficient ethanol producer, but can not utilize xylose. P. stipitis can produce ethanol from xylose, but under aerobic conditions. Xylose uptake is strongly inhibited by glucose, so it will always first utilize any available glucose and then switch to xylose. However, solutions have been described that solves some of these problems (Grootjen et al. 1991). Zygomycetes are very suitable for fermenting lignocellulosic hydrolysates. They are fairly tolerant to the inhibitors and can ferment glucose to ethanol at yields compara- ble to that of S. cerevisiae. The Zygomycetes also consume xylose, producing xylitol and biomass. The biomass could potentially be considered a valuable product. It has been suggested that Zygomycetes biomass might be used as fish feed. This would be a far more sustainable alternative to the controversial and ethically questionable fish feed used today (Lennartsson et al. 2011, Tacon & Metian 2009). Co-cultures of S. cerevisiae and P. stipitis immobilized in alginate beads has been used for fermenting enzymatic hydrolysate of steam-explosion pretreated wood chips by De Bari et al. (2004), who achieved a yield of 0.396 g ethanol per g total sugars, or 77 % of the theoretical maximum. Yeast immobilized in pellets of Zygomycetes biomass for the purpose of fermenting lignocellulosic hydrolyzates have been considered before, for instance, there is a

5 patent describing the use of such co-cultures in air-lift reactors (Edebo 2009).

2.2 Factors believed to inﬂuence pelletization

Pelleted growth of many different fungi have been described. Two review articles by Papagianni (2004) and Gibbs et al. (2000) discusses the many different factors that are believed to inﬂuence pellet formation. In particular, the effects of pH, oxygen tension, addition of polymers, surface active agents, growth rate and inoculum size are regarded as important factors and have also been discussed by Metz & Kossen (1977). A large number of different factors are known to cause or improve on pellet formation. Below is a summary of the most important factors and a short description of the effect they are believed to have on fungal mycelium.

2.2.1 Inoculum concentration

Many have noted the importance of the initial spore concentration. In the coagulative fungi, the spores first agglomerate into clusters and then germinate and grow to form a pellet. In the non-coagulative fungi, the spores do not agglomerate, but first germinate. The hyphae may then entangle and form pellets. If the biomass concentration is then too large, all hyphae might clump together forming larger irregular clumps or a single large mat. Higher spore concentrations therefore often prevent pellet formation, and each strain can be said to have a critical inoculum concentration. For non-coagulative fungi the critical inoculum concentration is 108–109 spores/l (Archer 1973, p. 789). The critical inoculum concentration depends on agitation level. van Suijdam et al. (1980) also states a critical inoculum concentration at 108–109 spores/l. Papagianni sets the critical inoculum concentration at 109 spores per liter for P. chrysogenum, A. niger, Streptomyces tendae and S. griseous, despite A. niger has been classified as a coagulative fungi (Papagianni 2004, p. 201). Liao et al. (2007) sets the critical inoculum concentration at 1.5×109 spores/l for R. oryzae, while byrne states it is between 4×105–107 for R. nigricans depending on agitation rate.

2.2.2 Agitation

Agitation of the culture medium is necessary for pellet formation. Pellets are often formed in shake ﬂask cultures and there is a general agreement that higher shaking frequency leads to smaller pellets (Gibbs et al. 2000, p. 29). Shaking frequencies between 100 and 220 rpm are normal and often lead to pellet formation. Pellets can also be formed if the cultures are shaken in a reciprocating fashion, rather than the more common rotational movement. There is an inverse relationship between the number of spores per pellet and the power input through agitation. This is generally interpreted to mean that the agitation breaks up larger and loose agglomerates so

6 that only ﬁrmer, highly intertwined agglomerates remain (Metz & Kossen 1977, p. 784–785). Pellets can also form in air lift reactors where the gentle agitation by the rising bubbles cause enough agitation for pellets to form. Basidiomycetes can form pellets under such circumstances and only forms dispersed mycelia under stronger agitation. Pelletization is also known to occur in stirred tank reactors, primarily from industrial production of penicillin by P. chrysogenum and was studied by Wittler (1983).

2.2.3 Calcium carbonate

Xia et al. (2011) observed that calcium carbonate induces pelleted growth of Mucor circinelloides. The growth medium was initially at pH 3 and very little growth occurred. After 18 h calcium carbonate was used to increase the pH to 5.3. This resulted in good pelletization. Liao et al. (2007) used a YPD-medium supplemented with calcium carbonate to form pellets with R. oryzae. Much attention has been focused on trying to determine what physio-chemical effect calcium carbonate has. It is possible that the higher pH and the pH-buffering effect is most important. The solid particles attach to fungal hyphae and are absorbed into pellets, suggesting a surface chemical effect. The particles might work as nucleation sites. Some calcium carbonate dissolves and so contributes to a higher calcium ion concentration. The ions may be important as ligands in surface bound proteins or they might change the dielectric constant of the medium, resulting in altered electrostatic interactions between cell surfaces.

2.2.4 Calcium concentration

The concentration of salts is believed to have an effect, and especially calcium has been studied. The interest for calcium partially comes from the observations of the positive influence of calcium carbonate. The evidence in the literature is conflicting. Xia et al. (2011) claimed that addition of calcium chloride did not contribute to pellet formation in M. circinelloides, but that the positive effect of calcium carbonate is due either to the pH-change or that the solid particles may serve as nucleation sites. The calcium concentration influences hyphal and cell morphology in Aspergillus niger. A medium without calcium gave a lower branching frequency and the biomass occurred as both pellets and clumps. Calcium in the form of 0.5 g/l CaCl2 increased branching frequency and gave better and more uniform pellets (Pera & Callieri 1997).

2.2.5 pH

Several researchers have studied the effect of pH on pelletization. Archer (1973, p. 239) found that a pH of 4.3–6.0 gave good pellets with Sclerotina fructigena. Metz & Kossen (1977) mentions that Aspergillus niger forms pellets at pH < 5 and

7 Penicillium chrysogenum at higher pH, up to 7.4. It is believed that pH is important for pellet formation because the pH changes the surface properties (surface charge) of the spores, making them agglomerate within certain pH-ranges. Zhou et al. (2000) reported that pH had an effect on the morphology of R. oryzae. Extremely low pH (less than 2.5) inhibited growth entirely and higher pH gave hairy pellets. Smooth uniform pellets were achieved at pH-values between 2.6 and 3.36, with an optimum at 3.05. Xia et al. (2011) found that a sudden increase in pH from 3 to 5.3 after 18 h of cultivation with Mucor circinelloides resulted in pellet formation, regardless of weather the pH adjustment was done using calcium carbonate or sodium hydroxide. The cell walls of most microorganisms are negatively charged at pH-values above 5.5, caused by deprotonation of proteins. This leads to a repulsive electrostatic force, which may be shielded by an increase in ionic strength or by bridging cells with calcium ions (Papagianni 2004, p. 200). Papagianni (2004, p. 227) concludes that pelletization is strongly inﬂuenced by pH and that in general, pellets occur at higher pH. pH is one of the most studied factors affecting pelletization, but the importance and the mechanism of action of pH on spore agglomer and particles of other cerealsation and pelletization is still unclear (Gibbs et al. 2000, p. 24).

2.2.6 Solid particles

Liao et al. (2007) reported that insoluble calcium carbonate induced pelletization in Rhizopus oryzae. Solid particles of rice flour also significantly contributed to pelletization of R. oryzae in the experiments by Liu et al. (2008). However, it is difficult to determine exactly what causes these observations. Is it the physical support of the particles, or the pH-buffering effect of the calcium carbonate? At lower pH, some of the CaCO3 will dissolve and calcium ions are known to affect pellet formation. In the case of rice, it is impossible to isolate the effect of the solid support, the nutritional value of the starch and the possible effect of the starch that dissolves in the medium.

Many other solid particles have been tested. Titanate (TiSiO4) have been reported to give small and highly reproducible pellets, while talc prevented pelletization of Aspergillus niger (Driouch et al. 2012). Wheat bran and other cereals have also been shown to induce or improve on pellet formation, but in this case, it might actually be caused by the phosphorous in the cereals (Papagianni 2004, p. 217). Fomina & Gadd (2002) studied the inﬂuence of three different clay minerals (ben- tonite, palygorskite and kaolinite) on the pelleted morphology of Cladosporium cla- dosporioides, C. herbarum and Humicola grisea. Clays affected the size, shape and structure of the pellets. The mineral particles became attached to the fungal hypae and was absorbed into the pellets. They also observed that clays generally reduced the size of pellets and that in some cases the clays reduced the branching frequency so that hairy pellets were formed. There is no known physio-chemical explanation for the effect of solid particles on

8 fungal morphology. Atomic force microscopy has been suggested as a good method for studies of this effect (Driouch et al. 2012, p. 469). It should be noted that the presence of solid particles in the medium is not necessary for pelletization (Martin & Bailey 1985).

2.2.7 Carbon dioxide

Carbon dioxide partial pressure, pCO2, influences fungal morphology and metabolism. Carbon dioxide has also been reported to affect chitin synthesis, exo-enzyme production and branching frequency. It also plays a role in the dimorphism of Mu- cor (Gibbs et al. 2000, p. 23). The influence of CO2 on filamentous fungi is under debate and there is no clear evidence in the literature that pelletization is affected. (Papagianni 2004, p. 225)

2.2.8 Nitrogen source and concentration

Byrne & Ward (1989a, p. 913) found that there was an inverse relationship between the peptone concentration and the number of pellets formed, using R. arrhizus. Op- timal peptone concentration was 5 g/l. By intermittent addition of ammonium sulfate so that the concentration is always higher than 0.1 g/l, the agglomeration of small ﬂocs of R. oryzae can be prohibited (Yu et al. 2007).

2.2.9 Carbon source and concentration

There is an inverse relationship between the glucose concentration and the number of pellets, but the average size/weight of the pellets increased with the higher sugar concentrations in the experiments done by Byrne & Ward (1989a). Glucose affected the amount of biomass formed and the size and number of the pellets, but did not affect the pelletization per se. Pelletization occurred at glucose concentrations between 5 g/l and 45 g/l. It is not known if different carbon sources affect pellet formation. Zygomycetes grows well on xylose, but pellet formation in xylose containing media have not been described earlier.

2.2.10 Surfactants

In coagulating fungi it is reasonable to believe surfactants should affect pelletization. Žnidaršicˇ et al. (1998) used 0.5 g/l Tween-80 in their medium for inducing pelleted growth of the non-coagulative Rhizopus nigricans. Their article does not mention why they included it or if they tested to exclude it.

9 2.2.11 Polymers

Addition of polymers to the culture medium can strongly affect pelletization. Car- bopol-934 and polyacrylate have a favorable inﬂuence on pellet formation (Metz & Kossen 1977) as well as rice starch (Liu et al. 2008). Solid maize particles prevents pelletization in Rhizopus arrhizus (Byrne & Ward 1989b).

2.2.12 Volume and ﬂask size

Shake flask cultivation is very common for studying pelletization. The shaking mo- tion creates a wave in the medium that propagates along the inner wall of the flask. The waves have different amplitude and period depending of the agitation rate, the size and shape of the flask and the volume of medium in the flask. It is likely that some wave motions are more favorable to pellet formation that others and that a high amplitude standing wave promotes formation of smooth pellets. This was confirmed in the experiments by Engel et al. (2011), who found that small pellets of R. oryzae formed when the liquid oscillated up on the flask wall. For higher shaking intensi- ties, the wave was out-of-phase, the liquid would not oscillate and dispersed mycelial growth predominated.

2.2.13 Metal ions

Zhou et al. (2000) claimed that the concentrations of Zn2+, Mg2+ and Fe2+ inﬂu- enced pelletization of R. oryzae and that the optimal concentrations were 4 ppm, 50 ppm and 100 ppb respectively. Of these, iron had the smallest effect on pelletization. They also mentioned that a Mn2+-concentration larger than 2 ppm prevented pelleted growth (Zhou et al. 2000, p. 789). Papagianni (2004, p. 221) discusses the importance of metal ions on the morphology and metabolism of A. niger, and concludes that manganese is essential for normal metabolism and morphology. She mentions an old study from 1966 that claimed that pelleted growth was prohibited by addition of 2 ppb Mn2+ to ferrocyanide treated molasses. Later studies however, have obtained pellets of A. niger at much higher concentrations, see for instance the paper by Driouch et al. (2012).

2.2.14 Chelating agents

Choudhary & Pirt (1965) studied the effect of several complex forming agents such as EDTA and ferrocyanide on the pelletization of A. niger. They found that either EDTA, CDTA or DTPA at 1.04 mM or ferrocyanide at 84 µM caused the organism to grow in the form of small pellets with a smooth exterior; such pellets remained discrete and dispersed in the medium. Higher concentrations of chelators prevented pelletization. The effect of chelators is attributed to the lower free metal ion concentration in the medium.

10 2.2.15 Ionic strength

If the agglomeration of spores or hyphae is caused by electrostatic forces due to surface charges, the ionic strength in the medium should have an effect. This is because electrostatic forces are attenuated in media with a higher dielectric constant.

2.2.16 Hydrophobic interaction

Gerin et al. (1993) observed that spores of Phanerocaete sporidium agglomerated after about 3 h, when the spores had begun to swell. This was attributed to chang- ing surface properties of the spores during the swelling. Especially the amount of polysaccharides increased. It was concluded that agglomeration results mainly from polysaccharide bridging and not from hydrophobic effects. Priegnitz et al. (2012) studied the surface properties of spores of A. niger using a mutant strain with altered surface hydrophobicity and surface charge. They found no differences in pelletization behaviour beween the two strains and concluded that initial aggregation of ungerminated spores do not contribute to pelletization. Papagianni (2004) mentions that factors inﬂuencing pelletization simultaneously increases cell wall hydrophobicity in A. niger.

2.2.17 Other factors

It is possible that temperature has an effect on pelletization. Xia et al. (2011) observed that the number of pellets and their size varied with temperature. Optimal temperature for pellet formation was close to the optimal growth temperature. Oxygen concentration has also been considered and Martin & Bailey (1985, p. 1504) writes that lower concentrations are favorable for pelletization. Caldwell & Trinci (1973) introduced a hyphal growth unit, G. It is the total hyphal length divided by the number of (viable) tips in a fungal mycelium. In essence, it is a number inversely proportional to branching frequency. It can be thought of as an analogy of a cell in single-celled organisms. When a hyphae has grown to the length G it ”divides” by forming a new branch. A highly branched and intertwined mycelium helps in developing dense, smooth pellets, so species with different G might be more or less suitable for pelleted growth. (Papagianni 2004, p. 195-196) van Suijdam et al. (1980, p. 212) pointed out that in industrial processes, the microorganism, strain and medium composition are normally ﬁxed due to the requirements of the process itself. So only a few factors may be changed to induce (or avoid) pellet formation. He mentions the inoculum concentration, polymer additives and shearing forces (agitation) as the three major variables that may be controlled. However, it is not necessary that the pellets are formed during the production fermentation. An inoculum already in the form of pellets may be used, perhaps reused from an earlier batch.

11 2.3 Co-cultures of fungi and yeasts

In nature filamentous fungi and yeasts often grow together on sugary substrates such as decaying fruits or cereals. In Indonesia co-cultures of Zygomycetes and yeasts are used to make tempeh, a traditional food made by fermentation of steamed soy beans and barley (Feng 2006). The Zygomycetes hydrolyzes the complex carbohydrates in the soy and barley into simple sugars that the yeast can then use. There are many examples of alcoholic beverages produced by fermenting rice starch or other cereals with co-cultures of Zygomycetes and yeast, or with A. oryzae and yeast (Hesseltine & Wang 1967, Tamang et al. 1988). One of the most important characteristics of these co-cultures is that the filamentous fungi excrete hydrolyzing enzymes, such as amylo-glucosidase, hydrolysing amy- lopectin, which yeasts are unable to do. The yeast can then use the resulting sugar and keeps the sugar concentration low, inducing continued enzyme excretion in the filamentous fungi (Nyman 2010).

2.4 Biocapsules van Suijdam et al. (1980, p. 211) suggested fungal pellets could be used ”as a support for whole cells”, but apparently never tried it. Peinado et al. (2005) first described how living yeast cells can be immobilized inside pellets of Penicillium chrysogenum and patented the technique. They claim that yeast cells will spontaneously immobilize if a co-culture is grown in a medium that fulfills a few key requirements: • A carbon source easily utilized by the filamentous mold and not the yeast. • A medium buffered at pH 7. • Agitation to facilitate pelletization and aeration. After a few days of cultivation spherical and hollow pellets with immobilized yeast cells inside was obtained. When these so-called biocapsules were placed in a rich medium with a carbon source suitable for the yeast, the pellets were colonized by the yeast. The mold eventually died, leaving capsules of dead fungal biomass filled with living yeast cells. A few more articles have since been published on these biocapsules (Martínez et al. 2011, Peinado et al. 2006, García-Martínez et al. 2012). Saccharomyces cerevisiae var. capensis was used in most of these studies. It is a yeast used in the production of sherry wines. After the main fermentation of the wine, this yeast floats to the surface and forms a thin biofilm, called flor and a second fermentation occurs. S. capensis produced better and smoother capsules than other non-flor forming yeast strains. Martínez et al. (2011) observed that the strong growth and colonization of yeast in the capsules caused the mold to die. They speculate that the yeast cells are attached to the mold hyphae by means of some sticky substance and that this close proximity between cells is responsible for the early death of the Penicillium. The article contains

12 electron micrographs (see ﬁgure 2.1) showing this interaction, but the evidence is not yet conclusive.

Figure 2.1: Transmission electron micrograph from the article by Martínez et al. (2011) depicting interaction and release of some substance (black arrows) between a yeast cell (Y) and a P. chrysogenum hyphae (Hy). Vesicles (white arrows) in the yeast cell was observed near the interaction point. Picture re- production granted by license from the publisher John Wiley and Sons.

There is convincing evidence of a cell-to-cell contact mediated mechanism killing one of the species in some binary co-cultures (Nissen & Arneborg 2003). The close proximity of cells and lack of space induces early death of Kluyveromyces thermotol- erans and Torulaspora delbrueckii when grown in co-cultures with Saccharomyces cerevisiae. The exact mechanism of this cell contact and early death is unknown. Fermentation of yeast and Penicillium in co-cultures, where the species are separated by a semipermeable dialysis diaphragm, was conducted to demonstrate that the death of Penicillium is indeed caused by the physical proximity of yeast cells on mold hyphae. The phenomenon is similar to that described by Nissen & Arneborg (2003). Biocapsules of Aspergillus and bacteria have also been studied for the purpose of bioremediation of aniline and azo-dye contaminated water (Yang et al. 2011, Zhang et al. 2011). The green microalgae Chlorella vulgaris was immobilized in pellets of Aspergillus and the biocapsules were used for waste water treatment. These biocapsules are also interesting for biodiesel production from the algal biomass that, thanks to the pelletization, is easily harvested (Zhou et al. 2012). Finally biocapsules have been used for production of hydrogen with Clostridium (Zhao et al. 2012).

2.5 Flocculation in yeasts

Yeast cells often have the ability to ﬂocculate, forming clumps of hundreds or thou- sands of cells that either sink to the bottom or ﬂoat to the surface. This have been

13 used for hundreds of years in beer-fermentation where flocculation is used as a simple way to separate the yeast after the fermentation. Consequently, flocculation has been studied extensively, but only recently has the underlying genetic mechanisms been understood (Verstrepen & Klis 2006, Van Mulders et al. 2009). Flocculation and adhesion to abiotic surfaces (such as plastic or agar) arises due to two different mechanisms. The first depends on lectin-like cell surface proteins called ”flocculins” or ”adhesins” which bind to sugar residues in the cell wall of neighbor- ing cells. This mechanism can be further divided into either the Flo1 or the NewFlo phenotypes. Flo1-dependent flocculation is interrupted by mannose in the medium and NewFlo-flocculation is inhibited by both glucose and mannose (Verstrepen et al. 2003). The second mechanism depends on an increased hydrophobicity in the cell wall. This is the case in S. cerevisiae var. capensis that is used in sherry wine fermentation. When the primary fermentation is completed, this yeast expresses highly hydrophobic proteins that causes the cells to float to the surface and form a floating wax-like biofilm, called ”flor”. The flocculins are encoded by several FLO-genes. The regulation of these is highly complex (Zhao & Bai 2009, Guo et al. 2000).

14 3 Materials and Methods

3.1 Microorganisms

Four Zygomycetes, all of the order Mucorales was used in this study. Two strains identified as Rhizomucor sp. RM3 and Rhizomucor sp. RM4 have been described previously by Wikandari et al. (2012). The strains have been deposited at the Cul- ture Collection of the University of Gothenburg as strains no. CCUG61146 and CCUG61147 respectively. Mucor indicus CCUG22424 was used in this study and this strain has also been described and studied earlier (Lennartsson et al. 2009, Mil- lati et al. 2005, Karimi et al. 2008). A fourth strain isolated from Indonesian tempeh and identified as Rhizopus sp. was also used. This strain has been studied earlier by Ferreira et al. (2012). Four yeast strains were used. Saccharomyces cerevisiae CBS8066 (Centraalbureau voor Schimmelcultures, Delft, the Netherlands) is a common laboratory strain. For instance, it has previously been encapsulated in alginate beads and used for fermenting lignocellulosic hydrolysates (Taherzadeh et al. 2001). It does not flocculate. Saccharomyces cerevisiae CCUG53310, a highly flocculating yeast that has been described in detail by Westman et al. (2012). A recombinant strain, S. cerevisiae CEN.PK 113-5D+pRS316-YAP1p-GFP was kindly provided by Valeria Mapelli, Chalmers Technical University, Gothenburg, Sweden. The strain expresses green fluorescent protein under oxidative stress. A naturally occurring mutant yeast strain, S. cerevisiae ade1−, with a dysfunctional ADE1-gene. The strain is adenine-auxotrofic and produces a red pigment. It will be referred to as red yeast. The Zygomycetes strains were maintained on PDA-agar plates and sub-cultured weekly to obtain fresh spores. The spores were harvested by adding 20 ml of sterile water and gently agitating the mycelium with a disposable cell spreader. The spore concentration was measured as OD660 using a standard curve obtained by manual counting of spores from each strain in a Bürker chamber. The GFP-expressing yeast strain was maintained on plates with an uracil-deficient defined medium. The other yeasts were cultivated on YED-plates.

3.2 Induction of pelleted growth

Several different media were tested in an effort to induce pelleted growth of the four fungi. Different media described by Karimi et al. (2008), Xia et al. (2011), citeas- nounyang, Liao et al. (2007), Žnidaršicˇ et al. (1998) and Zhou et al. (2000) were tested. The compositions of these media are listed in Appendix A. Martínez et al. (2011) demonstrated that if yeast cells are to be encapsulated in fungal pellets, it is important that the medium does not contain a carbon source utilized by the yeast, and that the medium is buffered. Because of this, a modiﬁed Liao-medium with the

15 glucose substituted for xylose (called Liaox-medium) was also tested. Yang-medium was tested with xylose. Spores were inoculated at the concentration used in the respective articles. M, indicus and Rhizopus sp. were also inoculated to very small spore concentrations since it was suspected that these strains have a very small critical inoculum concentration. Only fresh spores less than 10 days old were used.

3.3 Biocapsules

In an attempt to encapsulate and immobilize yeast cells inside fungal pellets, the following procedures were used. Spores of Rhizomucor sp. RM3 or RM4 was inoculated into 100 ml Liaox-medium in a 250 ml E-flask to a final concentration of 104–105 spores/ml. A loop-full of one of the yeast strains was added and the flasks were incubated in a thermostated waterbath with agitation (150–160 rpm). After 3–4 days, the pellets had grown to 4–5 mm diameter. A few pellets were then transferred to a new secondary medium. This secondary medium was used to make the yeast grow and colonize the pellet and stop further growth of the Rhizomucor. A medium containing 20 g/l sucrose and either 5 g/l yeast extract or 1x yeast nitrogen base was used.

3.4 Microscopy techniques

Light microscopes always have a very short focus depth. This makes it very difﬁcult to study large or three-dimensional objects. Pellets with a diameter of several mil- limeters can not be readily visualized in an ordinary light microscope, see ﬁgure 3.1. It is necessary to use more advanced methods to microscopy pellets.

3.4.1 Confocal and dark ﬁeld microscopy

Dark ﬁeld microscopy excludes any light that went through the specimen unscattered. Only scattered light reaches the ocular. The result is that the specimen under study is illuminated against a black background. This greatly enhances contrast and picture quality in microscopy. Microscopy samples are often stained with various pigments to increase contrast or to selectively make certain parts of the specimen visible. Several common stains, such as metylene blue, crystal violet and saffranin were tested in an effort to try to increase the visibility and contrast in light microscopy of Zygomycetes hyphae. In particular, a method was sought that stains yeast cells and Zygomycetes differently, so that the presence of yeast cells, immobilized inside fungal pellets, would be visible.

16 Figure 3.1: The short focus depth of a microscope allows only the equator of a pellet to be studied. Here, microscopic crystals of calcium carbonate can be seen absorbed in a pellet of Rhizomucor sp. RM3 grown in Liao-medium.

An alternative to staining is to use yeast strains that produces colored substances. An adenine auxotrofic strain with a mutation in the ADE1-gene is incapable of producing adenosine monophosphate. The precursor p-ribosylaminoimidazole then accu- mulates in the cell, giving it a pink or red color. This strain becomes red when grown aerobically in a medium with low, but non-zero, concentration of adenine. A recombinant yeast strain expressing green fluorescent protein was kindly provided by Chalmers technical university, Gothenburg. The GFP-gene sits in a plasmid with a promotor that is up-regulated under physiological stress. The strain is uracil-auxotrofic with a URA3-gene in the same plasmid, allowing selection in uracil- deficient media.

3.4.2 Focus stacking

Focus stacking is a digital image processing technique that can enhance the focus depth. Several photographs were taken at different focal depths. The images were then stacked using a computer program; ZereneStacker1. The computer program identiﬁes what parts of an image is in focus and removes the parts that are not. The partial images that are focused are then added together to form a new composite image. The resulting image consists of the parts of each of the several partially focused images that were in focus. Ideally, the entire image is then focused.

1http://zerenesystems.com/

17 ZereneStacker also have functions for manually retouching pictures. This can further greatly enhance the picture quality. However, since the method is manual, based on the operators subjective choices and non-reproducible, manual retouching was not used in this work. Only the PMax-algorithm was applied to a stack of images, normally consisting 4–7 individual photos.

3.4.3 Side-by-side merging

Several photographs may also be merged side-by-side, resulting in a larger and high- resolution picture. A number microphotographs were taken of the same specimen. Normally 12–20 individual photographs were taken in 100x enlargement. Very high resolution pictures were created by taking a large number (63, 80, 104) of photographs at 200x magniﬁcation and then merging them into one using Adobe Photo- shop CS3 and the ”photomerge” method.

3.4.4 Microtome sectioning

Methods for making microtome cross sections of pellets was developed based on descriptions by Wittler (1983, p. 44–45) and Bizukojc & Ledakowicz (2010, p. 44). Pellets were stained with either 25 % lactophenol cotton blue in phosphate buffered saline solution or with pyridinium iodide (200 µl 0.1% in 10 ml PBS) for one hour. The pellets were then ﬁxated in 4 % glutaraldehyde in PBS at room temperature for one hour. After ﬁxation, the pellets were dehydrated by treating them with a series of successively more and more hydrophobic solutions, see table 3.1.

Table 3.1: Dehydration of pellets by successively increasing hydrophobicity. solution duration temperature 1. 25 % ethanol 20 min 4◦C 2. 50 % ethanol 20 min -20◦C 3. 70 % ethanol 20 min -20◦C 4. 85 % ethanol 20 min -20◦C 5. 100 % ethanol 20 min -20◦C 6. 100 % ethanol 20 min -20◦C 7. xylene/ethanol (50/50) 30 min -20◦C 8. 100 % xylene 30 min -20◦C

The dehydrated pellets were then embedded in paraffin as described in table 3.2. The paraffin-embedded pellets were placed in aluminum casting molds and plastic cassettes were placed on top. Molten paraffin (60◦C) was then gently poured through the cassette, so that the pellets were completely embedded. The castings were stored at 4◦C overnight. It is also possible to embed pellets in agar. This is a very attractive alternative because it can be done at a lower temperature so that proteins do not denature. This technique

18 Table 3.2: Embedding of dehydrated pellets in histological paraffin. solution duration temperature xylene/paraffin (50/50) 60 min 60◦C 100 % paraffin 60 min 60◦C was therefore used for pellets co-cultivated with GFP-expressing yeast. A 1.5 % agar solution at 45◦C was poured in a petri dish and pellets were immediately submerged, see figure 3.2. After a few minutes cubes approximately 1 cm3 large can be cut from the gel with a scalpel so that each cube contains one pellet, see figures 3.4.4.

Figure 3.2: A petri dish with 1.5 % agar and submerged biocapsules of Rhizomucor sp. RM4 and GFP-yeast or CBS8066.

The gel-cubes are glued to plastic cassettes with warm agar solution. The cassettes with the attached agar cubes are then rapidly cooled in liquid nitrogen for 10–15 s. This makes the gel freeze instantly and very small ice-crystals are formed. The frozen agar is then stored at -20◦C. Rapid freezing is necessary, a slow refrigeration would form large ice-crystals that would fracture the pellet. If the agar is submerged in liquid nitrogen for a longer time than 15 s it fractures. The paraffin preparations were cut in a microtome into circa 5–30 µm thin sections. The thin layers of paraffin were gently laid on the surface of a water bath thermostated at 42◦C, so that they would unfold. They were then mounted on glass slides. To prevent the sections from detaching from the glass slides during de-paraffination and rehydration, so-called subbed slides were used. The glass surface is treated with a dilute solution of gelatin (1 g/l) and chromium potassium sulphate, CrKSO4, 0.1 g/l. The chromium(III)-ions form coordinate bonds with electronegative atoms in the

19 Figure 3.3: Biocapsules of Rhizomucor sp. RM4 and different yeast strains embedded in paraffin (left) or agar (right) and mounted on green plastic cassettes. protein, forming a cross-linked polymeric adhesive (Kiernan 1999). After the paraffin sections had dried on the the slides, the paraffin was removed by careful application of xylene, which readily dissolved paraffin. The agar cubes were kept refrigerated by applying a cryo-spray while they were sectioned in the microtome. The sections were mounted on untreated glass slides and the agar allowed to melt. A slide with an agar section is depicted in figure 3.4(a).

Figure 3.4: In picture (a): A slide with a microtome section of a pellet embedded in agar. To the right (b): Two subbed slides with several parafﬁn sections.

20 3.5 Multi-factorial experiment

Many different factors are known to inﬂuence pelletization, but the literature is to a large extent descriptive, subjective and qualitative. Many experiments have been relatively simple and often neglect to take interaction effect into account, even when these are highly likely to exist. It is very hard to draw any conclusions about what causes pelletization (Gibbs et al. 2000, p. 22). Despite the large interest in pellet formation, very few studies have used modern statistical experimental designs, Liao et al. (2007) and Liu et al. (2008) being two notable examples. In an effort to try to understand how different factors inﬂuence pelletization in Zy- gomycetes, and especially under such conditions that allow immobilization of yeast cells inside the pellets, a multifactorial experiment was performed. A Central Com- posite Design with eight factors, face centered axial points and 8 center points was chosen as the experimental design. The design table and raw data is included in Ap- pendix B. The eight factors were: temperature, agitation intensity, calcium concentration, a surfactant (tween 80), inoculum concentration, addition of solid particles (cellulose), pH and the volume of medium. The factors were tested at three levels as indicated in table 3.3. Factor low level center level high level Temperature 25◦C 30◦C 35◦C Agitation 120 rpm 155 rpm 190 rpm CaCl2 conc 3.4 mg/l 2.5 g/l 5 g/l Tween 80 0 g/l 0.25 g/l 0.5 g/l Log inoculum conc 2.3 ml−1 3.3 ml−1 4.3 ml−1 Cellulose particles 0 g/l 1g/l 2 g/l pH 4 5 6 Volume 25 ml 38 ml 50 ml

Table 3.3: The eight different factors and their levels in the factorial experimental design.

The experiments were carried out in identical 100 ml wide neck borosilicate glass Erlenmayer-flasks (Fischer scientific FB33139). (8−2) The 2V -design allows estimation of the effect of each of the eight factors, as well as two-factor interactions and quadratic terms (Montgomery 2004, p. 331). The experiment is suitable to determine what factors have an effect on pelletization, and it should be possible to find the optimal conditions for inducing pelletization. Interac- tions between different factors might reveal something about the underlying physio- chemical phenomena causing pelleted growth. The factorial design was created using Minitab 15. The full design table contained 64 corner points, 16 are face centered axial points and 8 are center points, for a total of 88 runs. After four days of cultivation the flasks were cooled to 4◦C and a sample were col- lected for HPLC-analysis. The number of pellets in each flask was counted manually

21 and the mean diameter was estimated. The pH was also measured, at room temperature. The HPLC-analysis gave data on the concentration of xylose remaining in the ﬂasks and how much of a few different metabolites was produced.

3.6 Logistic regression

Instead of using the number of formed pellets as the response, it is possible to use a binary response variable that takes the value ”1” when pellets are formed and ”0” otherwise. This qualitative dependent variable can then be used in a binary multiple logistic regression. There is one article by Liu et al. (2008) that describes exactly one such experiment, examining the effects of several factors on the pelletization of Rhizopus oryzae. In generalized linear models the regression equation

0 µ = β0 + β1x1 + β2x2 + ... + βkxk + = x β (3.1)

0 is used, where the x β is often referred to as the linear predictor. The βi are the regression coefﬁcients, and xi is the level of factor i. The relationship between the response mean µ and the linear predictor may be more complex than the identity stated in equation 3.1. The equation describing the relationship between the two is called a link function. In binary logistic regression the logit function, in equation 3.2, is usually used as link function (Montgomery 2004, p. 563-567). µ ln = x0β (3.2) 1 − µ Which leads to the model 1 µ = (3.3) 1 + e−x0β

The response variable takes on the values 0 or 1 depending on weather pellets form or not. The logistic regression may then be interpreted to be the probability of pellet formation for any given treatment. Using the same data as in the response surface design, a logistic regression was attempted using Minitab.

3.7 Bioreactor fermentations

To evaluate the performance of pellets in fermentations a number of batch reactor experiments were carried out. A 2 l reactor with 1.5 l working volume was used. The reactor was equipped with a rotary impeller, thermometer, pH-meter, cooler and a CO2-monitor. A control unit was used to continuously monitor temperature and pH and the unit could autonomously add NaOH-solution and control cooling and heating of the reactor in order to maintain constant fermentation conditions.

22 Samples were taken regularly during the fermentations and analysed by HPLC. The HPLC-system consisted of an Alliance Waters 2695 with an Aminex HPX-87H- column and equipped with a refractive index detector 410 and UV-absorbance detector 2487. The eluent was 5 mM H2SO4 at a ﬂow rate of 0.6 ml/min. The HPLC allows for analyses of glucose, ethanol, glycerol, acetic acid, lactic acid, pyruvic acid, succhinic acid, xylose, furfural, and 5-hydroxymethyl-furfural.

3.7.1 Fermentation of Sues medium

Medium according to Sues was autoclaved in a reactor and pellets of either Rhizomu- cor sp. RM3 or RM4 was inoculated. The fermentation was carried out at 30.0◦C. The pH was not allowed to drop lower than 5.5 by automatic addition of NaOH. HPLC-samples were taken regularly for 32 h.

3.7.2 Fermentation with limonene

It was speculated that the reduced diffusion rate inside fungal pellets might protect yeast cells from toxins. Perhaps yeast cells could survive and remain metabolically active inside pellets when these were submerged in a broth containing an inhibitor. Limonene is a strong inhibitor of yeast growth. The effect of limonene and other inhibitors on Zygomycetes and yeasts have been studied earlier by Lennartsson et al. (2009). Biocapsules of Rhizomucor sp. RM4 and S. cerevisiae CBS8066 were inoculated into Sues medium supplemented with 0.25 % and 0.33 % limonene in a CSTR, as described above. The fermentations were carried out with the stirrer at 300 rpm for 32 hours and samples were taken repeatedly for HPLC-analysis. The higher stirring speed was neccessary to keep the limonene in suspension.

3.7.3 Fermentation of lignocellulosic hydrolyzate

A dilute sulfuric acid hydrolysate of spruce with pH 2 was neutralized by addition of solid Ca(OH)2 powder until the pH reached 5.5. A precipitate of CaSO4 formed and was removed by centrifugation and decantation. The hydrolysate was diluted with ultra pure water to 60 % v/v. The hydrolysate was used as carbon source only. Ammonium sulfate, salts, trace ele- ments and vitamins were added to a ﬁnal concentration equal to that of Sues medium. Antifoam was also added. The fermentation was carried out with the stirrer set to 200 rpm, the temperature to 30◦C and the pH was maintained at 5.5. HPLC-samples were taken at regular intervals.

23 3.7.4 Ethanol evaporation

Some ethanol evaporates during the fermentation in the CSTR. The evaporation rate was determined by using an ethanol solution in a reactor under the same conditions as during the fermentations. A 6 g/l ethanol solution was run in a reactor for 33 h at 30◦C and 200 rpm. The aeration rate was also the same as during the fermentations. Samples were taken repeatedly and the ethanol concentration over time was plotted in in a diagram. A linear regression can then be used to ﬁnd an expression for the evaporation rate.

dc E(c) = · c h−1 (3.4) dt Where c is the ethanol concentration. Of course, when c is zero, is follows that the evaporation rate is zero. It must be stressed that this linear equation is only valid for small concentrations, where the solution is approximately ideal.

24 4 Results

4.1 Formation of pellets

The medium described by Liao gave excellent results with both strains of Rhizomu- cor. The glucose could be substituted for xylose with no adverse effects on pelletization. These media give uniform spherical pellets of highly intertwined mycelium. The pellets are highly resistant to mechanical forces and keep their shape for a long time. If xylose is used, yeast cells can be co-cultivated with the pellets. Pellet formation in these media is perfectly reproducible, giving practically identical results every time. A representative pellet is depicted in figure 4.2. It was difficult to obtain pellets of Mucor indicus. It was observed that this fungi has a much lower branching frequency than the others. The long unbranched hyphae do not entangle but grow radially out from the centre of the pellet, see figure 4.1. This prevents the formation of dense, highly intertwined pellets. The medium that gave best results with M. indicus was the medium described by Yang et al. (1995), supplemented with trace metals. However, even in this medium, the pellets were loose, hairy and the results not fully reproducible. Yang-medium was also suitable for inducing pelleted growth in Rhizopus sp. The medium is nutrient poor and deficient in several metal ions. Addition of trace metals (5–10 ml/l) resulted in a shorter lag phase, higher biomass yield and more pellets.

Figure 4.1: Hairy pellets of M. indicus grown in Yang medium. Hyphae do not intertwine because of the low branching frequency, preventing formation of proper pellets.

In runs 41, 42, 46 and 50 of the factorial experiment extremely large pellets formed. These were very different from normal pellets. They had a perfectly smooth surface and very low density. These pellets would not support their own weight but would

25 Figure 4.2: A stereoscope picture of a pellet of Rhizomucor grown in Liaox-medium. The calcium carbonate is absorbed into the pellet and a perfectly spherical, very dense pellet with a smooth surface is obtained.

flatten if removed from the medium and placed on a flat surface. These pellets ap- peared gray and not white as normal dense pellets. This kind of pellets will be called ”type II”-pellets. A representative picture of a few type II-pellets are depicted in figure 3(c).

4.2 Response surface optimization results

The data from the multifactorial experiment was analyzed using Minitab 15. A response surface regression was fitted to the square root of the number of pellets formed. A square root transformation was used to reduce the heteroscedasticity, as described in (Montgomery 2004, p. 80-82). The model was then reduced until only factors significant at the α = 0.2 level remained. The hierarchy principle was re- spected. The resulting regression coefficients, student’s T and p-values are presented in table 4.1. The residual standard deviation was s = 3.497, R2 = 54.6 % and adjusted R2 was 47.3 % The corresponding ANOVA table is presented in table 4.2. To evaluate the adequacy of this model, the residual plots were studied. An adequate model have homoscedastic and independent residuals. The residuals should be normally distributed. The residual plots for the model is given in figure 4.4. The residual plot of standardized residuals versus fitted value, in the upper right corner of figure 4.4, reveals a pattern. A number of points form a straight line. This pattern arises because the quadratic regression predicts a negative number of pellets

26 (a) (b)

Figure 4.3: The results from four different runs in the factorial experiment. (a) Run 39, large uniform pellets; (b) Run 41, very large pellets of type II; (c) Run 55, Dispersed mycelium; and, (d) Run 70, irregular pellets and clumps in many cases. But since a negative number of pellets can not form in reality, this model gives inaccurate predictions and is inadequate. In fact, a quadratic regression will always be unable to correctly model the pellet formation in this factor space. However, near the optimum, a quadratic response surface should be adequate. If the model is restricted to only fit those parts of the factor space that does result in a positive number of pellets, a response surface might adequately model the region close to the optimum. We loose a lot of degrees of freedom for the error so the precision will be small, but the model is more likely accurate. A new response surface was fitted to the square root of the number of pellets formed, and only including those runs, in which pellets were formed. Runs that resulted in zero pellets were replaced by ”missing value” in Minitab. The model was reduced until only factors significant at a p < 0.2 level was included.

27 Table 4.1: Coefﬁcients, Student’s T and p-values for a response surface ﬁtted to the square root of the number of pellets. Term Coef SE Coef T p Constant 8.6783 0.7895 10.993 0.000 Agitation -0.1595 0.4305 -0.371 0.712 Calcium 0.8620 0.4305 2.002 0.049 Surfactant 0.1159 0.4305 0.269 0.789 Spore 0.7357 0.4305 1.709 0.092 Particles -0.4126 0.4305 -0.958 0.341 pH 0.0816 0.4340 0.188 0.851 Volume -0.0233 0.4305 -0.054 0.957 Agitation*Agitation 4.1220 2.0421 2.019 0.047 Particles*Particles -5.7252 2.0421 -2.804 0.006 Volume*Volume -5.7910 2.0421 -2.836 0.006 Calcium*Spore 0.5988 0.4372 1.370 0.175 Surfactant*pH 0.7791 0.4372 1.782 0.079

Table 4.2: Analysis of Variance for the square root of the number of pellets. Source DF Seq SS Adj SS Adj MS F p Regression 12 1090.01 1090.01 90.83 7.43 0.000 Linear 7 100.43 99.03 14.15 1.16 0.338 Square 3 927.77 927.77 309.26 25.29 0.000 Interaction 2 61.80 61.80 30.90 2.53 0.087 Residual Error 74 905.08 905.08 12.23 Lack-of-Fit 65 791.86 791.86 12.18 0.97 0.576 Pure Error 9 113.22 113.22 12.58 Total 86 1995.08

The hierarchy principle was obeyed. This new ”positive” response surface had coef- ficient as listed in table 4.3. The residual standard deviation for this regression is s = 2.098, R2 = 90.2 % and adjusted R2 = 83.2 %. The Analysis of Variance for positive is presented in table 4.4. This response surface fits the data and shows that the quadratic terms have a very large effect on the number of pellets formed. The pH, volume of medium and inoculum size have large effects. More surprisingly, the surfactant and the cellulose particles also seem to influence pellet formation. This model does seem adequate and trustworthy, but because of the few data points used, care must be taking when interpreting the results.

28 Figure 4.4: Residual plot for a response surface ﬁtted to the square root of the number of pellets. Model reduced to include factors sig- niﬁcant at p < 0.2.

Table 4.3: Response surface coefﬁcients ﬁtted only to strictly positive responses. Term Coef SE Coef T p Constant 13.3115 0.6634 20.065 0.000 Temp -2.7965 0.8153 -3.430 0.003 Agitation 3.5830 0.6399 5.599 0.000 Calcium -1.0568 0.6500 -1.626 0.119 Surfactant -0.0565 0.5531 -0.102 0.920 Spore 2.0281 0.6463 3.138 0.005 Particles -0.0434 0.6122 -0.071 0.944 pH 0.2263 0.5961 0.380 0.708 Volume -1.0982 0.5331 -2.060 0.052 Temp*Temp -3.9848 1.6251 -2.452 0.023 Calcium*Calcium -9.8115 1.6251 -6.038 0.000 Surfactant*Surfactant -8.9407 1.6251 -5.502 0.000 Spore*Spore -7.5936 2.2933 -3.311 0.003 Particles*Particles -11.3796 1.6251 -7.003 0.000 pH*pH 48.9588 5.2696 9.291 0.000 Volume*Volume -11.4455 1.6251 -7.043 0.000

29 Table 4.4: ANOVA table for the response surface using only runs with pellets. Source DF Seq SS Adj SS Adj MS F p Regression 15 850.859 850.859 56.7239 12.89 0.000 Linear 8 351.810 234.341 29.2927 6.66 0.000 Square 7 499.049 499.049 71.2927 16.20 0.000 Residual Error 21 92.429 92.429 4.4014 Lack-of-Fit 14 27.763 27.763 1.9830 0.21 0.993 Pure Error 7 64.666 64.666 9.2380 Total 36 943.287

Figure 4.5: Residual plot for restricted model, using only data from positive experimental runs. The residuals are normally distributed, homoscedastic and independent.

4.3 Logistic regression results

A logistic regression solves several of the problems experienced in the response surface regressions. All available data may be used to find a model estimating the probability of pelletization for different treatments. Using Minitab 15 the following factors were found to have a significant effect on pellet formation. Temperature, Agitation, Calcium, Particles*Particles and pH*pH. The regression coeffients, p-values and odds ratios are presented in table 4.5. Only significant factors were included in the model. Liu et al. (2008) used p < 0.2 as their criterion for significance. The quadratic Particle-term was included, without it no fit model could be found. In this model the hierarchy principle is violated. This can be motivated by noting that the linear terms for pH and Particles had very small coefficients in the response surface, and that the optimal level is very close to the center point, giving negligible linear terms. The Log-Likelihood is -29,492, indicating a good fit. Test that all slopes are zero:

30 Table 4.5: Logistic regression table Predictor Coef SE Coef Z p Odds ratio Constant 2.7457 0.8404 3.27 0.001 Temp 1.4380 0.4619 3.11 0.002 4.21 Agitation -1.2059 0.4420 -2.73 0.006 0.30 Calcium -1.2059 0.4420 2.73 0.006 3.34 Particles*Particles -1.7425 1.3747 -1.27 0.205 0.18 pH*pH -2.8925 1.3369 -2.16 0.030 0.06

G = 59.675, DF = 5, p-Value = 0.000. The very low p-value shows that the factors included in the model does have an effect on pelletization. Three different goodness-of-ﬁt tests have large p-values indicating that the model is adequate.

Table 4.6: Three Goodness-of-Fit tests for the logistic regression Method Chi-Square DF p Pearson 5.57534 11 0.900 Deviance 7.24260 11 0.779 Hosmer-Lemeshow 3.16468 7 0.869

This logistic regression does ﬁt the data and the probability of pellet formation can now be calculated as 1 P (pellet) = (4.1) 1 + e−x0β Where 0 2 2 x β = β0 + β1x1 + β2x2 + β3x3 + β4x4 + β5x5 (4.2)

The βi being the regression coefﬁcients for factor i.

Table 4.7: Regression coefﬁcients for the model i Factor Coefﬁcient 0 Constant 2.7457 1 Temperature 1.4380 2 Agitation -1.2059 3 Calcium -1.2059 4 Particles*Particles -1.7425 5 pH*pH -2.8925

And xi is the level of factor i in coded units.

4.4 Substance 243

In the HPLC-samples from the factorial experiment an unknown metabolite was discovered. It eluted at 24.3 minutes and was therefore called ”substance 243”.

31 This substance was produced in the base medium used in the factorial experiment and in Yang-medium but not in richer media such as Liao, suggesting that the substance is produced under physiological stress. Both Mucor indicus and Rhizopus sp. produced it. Signiﬁcantly more was produced at 35◦C, compared to 25 and 30◦C (p < 0.01). The other factors in the factorial experiment did not have a signiﬁcant effect on the production of substance 243. Mucor indicus produced the substance in Yang-medium, but not if the medium was supplemented with 10 ml/l trace metals, further suggesting that the metabolite is produced during stress. The substance may be a substrate for an enzyme requiring one of the trace metals as co-factor. By comparing the detector responses for the refractive index and the UV-absorbance detectors of the HPLC, a RI/UV-number can be calculated for each peak in the chro- matograms. This way it could be determined that the unknown substance is not a simple alcohol or a simple carboxylic acid. The RI/UV-number of substance 243 was much smaller than any of the other (known) metabolites indicating that it has a very high absorbance for UV-light, strongly suggesting that it contains (conjugated) double-bonds, aromatic structures or is di-carboxylic acid. Fumaric acid, succhinic acid and similar compounds can be ruled out as these elute much earlier.

4.5 Biocapsules

The red adenine-auxotrofic yeast could not be seen inside pellets of Rhizomucor sp. RM4, but the medium did acquire a reddish color, see figure 4.6. A red discoloration, most likely due to live yeast cells, could be seen in clumps of fungal biomass that grew attached to the inside wall of the E-flasks, see figure 4.7. Since it was growing above the surface and medium was repeatedly splashing over this biomass clumps, ideal conditions (high oxygen/low adenine concentration) for the formation of the red pigment occurred in these clumps. Biocapsules with CCUG53310 could be obtained with either Rhizomucor strain. If the biocapsules were transferred to a secondary medium containing sucrose, the yeast would grow and small flocs of yeast would grow embedded in the mycelium. A secondary medium containing sucrose and yeast extract did not inhibit further growth of the filamentous fungus, so very large biocapsules approximately 10-14 mm i diameter were obtained, see figure 4.11. If biocapsules with CCUG53310 were grown in a secondary medium containing sucrose and yeast nirogen base without aminoacids, the filamentous fungus would soon stop growing while the yeast grew well and colonized the mycelium. Loose flocs of yeast would grow on the surface of the biocapsules.

32 Figure 4.6: Biocapsules of Rhizomucor sp. RM4 and red yeast, incubated in YED broth. The medium and the capsules are pink because of the red pigment produced by the yeast, demonstrating that the fungi can live together.

Figure 4.7: A clump of Rhizomucor sp. RM4 biomass from a co-culture with red yeast. The red discoloration is due to living yeast cells embedded in the fungal mycelium, demonstrating that yeast cells can live embedded in Zygomycetes biomass.

33 4.6 Results of Microscopy

Dark ﬁeld microscopy gives very good pictures and is especially suitable for Zy- gomycetes. The white mycelium has a high contrast against the black background. Since pellets are very large compared to the area covered in a microscope it was impossible to photograph an entire pellet in one picture. Several images may be merged into one, covering a larger area. This technique also allows the construction of photographs with very high resolution. It seems there is no simple method that stains Zygomycetes and yeasts differently. Most staining techniques have been developed for histological sections or specimens containing only one microbiological species. It was found that lactophenol cotton blue gives a clear blue stain to Zygomycetes cell wall. This greatly increased the visibility and made the highly intertwined mycelium easier to study. A stained pellet is depicted in ﬁgure 4.8. The contents of lactophenol cotton blue is given in Appendix A, on page 62.

Figure 4.8: A clump of Rhizopus sp. stained with lactophenol cotton blue. The small growth unit/high branching frequency is good for forming compact and smooth pellets. Staining gives higher contrast. This irregular clump is from run 59 in the factorial experiment.

Focus stacking proved to be a valuable method for obtaining images with a deeper focus depth. When pellets of Rhizomucor sp. RM4 grown with the GFP-expressing yeast was examined in ﬂuorescence microscope, it was discovered that Zygomycetes are highly ﬂuorescent and glows brightly in green when illuminated with UV-light. Because of

34 Figure 4.9: The inside of a pellet of Rhizomucor. The pellet was embedded in agar and sectioned in a microtome to approximately 100 µm thickness. The image shows the pellet’s hollow interior and the dense fungal mycelium viewed from the inside. this GFP-expressing yeast is probably not a suitable way to detect yeast encapsulated in fungal pellets.

4.6.1 Focus stacking results

It was found that ZereneStacker’s PMax-algorithm gave good results with dark ﬁeld microscopy images. Stacking makes it possible to see the three-dimensional surface of the pellets. It becomes possible to study the outer layer of growing hyphae pro- truding from the surface, see ﬁgure 4.16.

4.7 Results from bioreactor experiments

4.7.1 Ethanol evaporation rate

The evaporation rate was linear. If we assume that the evaporation rate also increases linearly with the ethanol concentration for small concentrations, we ﬁnd that the evaporation rate, E(c) is dc E(c) = · c = 4.21 × 10−3 · c h−1 (4.3) dt This means that the evaporation rate is very small and does not signiﬁcantly affect the data from the reactor experiments. The data has therefore not been corrected for evaporation.

35 Figure 4.10: A very high resolution picture of a microtome cross section of a parafﬁn-embedded Rhizomucor pellet. The diameter was 4 mm. The resolution is 26 megapixels. The image was obtained by merging 63 individual photographs taken at 200x magniﬁcation.

4.7.2 Fermentation of Sues medium with pellets

Pellets of Rhizomucor sp. RM3 were inoculated into a CSTR with medium according to Sues (2005). Samples were analysed by HPLC. The results are presented in figure 4.18. There was no lag time and glucose was fermented to ethanol in the first 13 hours. The yield of ethanol was 0.42 g/g, or 82 % of the theoretical maximum. Maximal ethanol productivity was 0.84 g/l·h. A significant amount of glycerol was formed, reducing the yield of ethanol. Once the glucose was depleted, the fungi started to utilize xylose and glycerol simultaneously which resulted in a strong biomass buildup. The uptake rate of xylose was clearly constant over time, indicating oxygen was the limiting nutrient. Xylose was utilized at a rate of 0.08 g/l·h. The pellets did not fragment but kept their morphology and grew from approximately 4 to 7 mm in diameter. Some pellets became attached to the impeller and baffles but the viscosity remained low as there were practically no freely suspended hyphae. An identical experiment was performed with pellets of Rhizomucor sp. RM4. The ethanol yield and productivity was 0.42 g/g and 0.8 g/l·h. Glycerol was not reassim- ilated and xylose was utilized at a rate of 0.14 g/l·h, see figure 4.19.

36 Figure 4.11: A cross section of a biocapsule of Rhizomucor sp. RM4 and CCUG53310. The dense inner pellet formed in the Liao- medium is clearly visible. The pellet continued to grow in the secondary medium, forming an outer shell, containing small ﬂocs of immobilized yeast. The diameter is 14 mm.

Figure 4.12: Detail of the previous image. Flocs of immobilized yeast inside a biocapsule of Rhizomucor sp. RM3.

37 Figure 4.13: Light microscopy image of a yeast ﬂoc inside a pellet of Rhi- zomucor. Microtome section approximately 15 µm thick.

Figure 4.14: A high resolution image of a microtome cross-section of a biocapsule of Rhizomucor sp. RM3 and the ﬂocculating yeast CCUG53310. Small ﬂocs of yeast cells are embedded and immobilized in the fungal mycelium.

38 Figure 4.15: Detail of the previous picture. Flocs of yeast are attached to the fungal hyphae and are immobilized in the biocapsule.

Figure 4.16: The surface of a pellet of Rhizopus sp. The pellet was grown in Yang medium and stained with lactophenol cotton-blue. The image was made by stacking ﬁve photos taken with slightly different focus.

4.7.3 Fermentations with limonene

The two reactors containing 0.25 and 0.33 % limonene are depicted in ﬁgure 4.20. Limonene at 0.25 % caused a very long lag time of approximately 24 h, after which 39 Figure 4.17: The ethanol concentration decreased linearly due to evaporation.

Figure 4.18: Glucose was rapidly fermented to ethanol with glycerol as the main byproduct. Linear and slow utilization of xylose depending on the oxygen transfer rate. exponential growth occurred, see ﬁgure 4.21. 0.33 % limonene caused a lag time longer than 32 h.

40 Figure 4.19: Rhizomucor sp. RM4 did not utilize glycerol and assimilated xylose at a faster rate than RM3.

Figure 4.20: The reactors containing limonene after 32 h fermentation. The reactor to the left contained 0.25 % limonene and the pellets are metabolically active. Bubbles of CO2 inside the biocapsules make them ﬂoat. The medium contained free- living yeast cells. The reactor to the right contained 0.33 % limonene and the biocapsules were still not active after 32 h and sank to the bottom. At this limonene concentration yeast cells can not survive freely suspended.

41 Figure 4.21: Fermentation with 0.25 % limonene. After a long lag time the exponential growth begins.

42 A few pellets from each reactor was washed twice with sterile water and placed on YED-agar and incubated at 30 ◦C for 48 h. Both Rhizomucor and yeast grew very well demonstrating that they had survived for 32 h in 0.33 % limonene, see ﬁgure 4.22 This suggests that fungal pellets might protect yeast cells in an environment that does not allow growth of freely suspended cells.

Figure 4.22: A few pellets from the limonene experiment was incubated on YED-agar plates. Both yeast and Rhizomucor grew well demonstrating that the fungi can live together and that yeast can survive inside pellets when these are submerged in a medium containing limonene at a concentration not support- ing free-living yeast cells.

4.7.4 Fermentation of hydrolysate

Soft wood hydrolysate was fermented with biocapsules of Rhizomucor. sp RM4 and CBS8066. The yeast was not immobilised and probably died soon after inoculation. After a 40 h lag time growth was exponential for 20 h until the glucose was depleted, see figure 4.23. Consumption of other sugars like arabinose and galactose must have occurred during glucose utilization, as the ethanol/glucose yield was higher than the- oretically possible. Xylose was utilized slowly and growth was inhibited by the limited oxygen supply. There was a large buildup of biomass during the linear phase that lasted 40 h until the xylose was depleted. The biomass was attached to baffles and instrumentation in the reactor, see figure 4.25 on page 45. It is clear that pelleted growth reduces the viscosity of the broth, but does not prevent the biomass from attaching to baffles. During the lag phase there was a substantial degradation of both furfural and 5- hydroxymethyl-furfural. After a 25 h lag phase, the degradation was exponential until the concentrations were practically zero. Growth of biomass started when there

43 was no remaining furfural and the hydroxymethyl-furfural concentration had been reduced to 0.35 g/l, half the initial concentration, see ﬁgure 4.24.

Figure 4.23: Consumption of glucose and other sugars yields ethanol and glycerol and products. Note the very long lag phase.

Figure 4.24: Zygomycetes can degrade furfural and 5-hydroxymethyl- furfural. This happens before the exponential growth phase.

44 Figure 4.25: Biomass attached to bafﬂes and intruments after fermentation of soft wood hydrolysate with pellets of Rhizomucor.

45 5 Discussion

5.1 Pelletization

Fungal pelletization is a very complex and poorly understood phenomenon. Despite the considerable effort that has been invested in studying pellets of the industrially important species Aspergillus and Penicillium the underlying physical and chemical mechanism that causes pelletization remains largely unknown (Gibbs et al. 2000, Papagianni 2004). Of the many factors believed to influence pelletization, inoculum concentration is probably the most important. An initial spore concentration smaller than approximately 108 spores/l is essential. The pH should not be allowed to drop, but a buffered medium should be used. Nutrient poor media are better for pellet formation than rich media. Calcium carbonate contributes to the formation of smooth and uniform pellets and higher agitation rates reduces the size of the pellets. These findings are well established and our results confirm them for Zygomycetes. There doesn’t seem to be any correlation between pelletization behavior and phylogeny. Remotely related species are sensitive to the same physical conditions when forming pellets and closely related Zygomycetes form pellets under very different conditions. In particular, these observations makes the classification of fungi into coagulative and non-coagulative species somewhat dubious. The experiments by Priegnitz et al. (2012) casts serious doubts on the coagulation theory. They used a mutant strain of the coagulative A. niger with clearly different surface properties but could not find any differences in the pellets obtained. The pelleted morphology have several advantages when it comes to mass transport and viscosity in stirred tank reactors and allows for a very convenient biomass harvesting. This is potentially of such great value in biotechnological processes that more research is warranted. More effort must be made in trying to establish a general theory explaining pelletization. The review articles by Papagianni (2004) and Gibbs et al. (2000) are excellent and discusses most things of importance when it comes to fungal pellets. However, they make no distiction between the ”primitive” Zygomycetes and the higher fungi. Pellets of Penicillium are routinely used in industrial scale fermentation with good results. It should be possible to use Zygomycetes in the form of pellets for production of fumaric acid and ethanol.

5.2 Multi-factorial experiment

Response surface methodology is based on an underlying assumption that the curva- ture of the response surface is maximally quadratic. In this case, a response surface can never adequately model the experimental results, because it will predict negative responses in many treatments. Response surfaces are inherently unable to correctly model a factor space where pelletization does not occur. It is possible to predict the

46 number of pellets that are formed, if a response surface is fitted only to strictly positive responses. However, the statistical results does show that there is a minimum close to pH 5 (p < 0.005). At this pH, the cell walls have a very small surface charge. It seems that a surface charge is beneficial for pellet formation. It is well known that solid particles are good for pelletization, and this seems to be the case also for cellulose particles (p < 0.005). It was observed that the particles were absorbed into the pellets, perhaps being attached to lectin-like proteins. The optimal cellulose concentration was 1 g/l. Response surfaces are inherently unable to correctly model a factor space where pelletization does not occur. It is possible to predict the number of pellets that are formed, if a response surface is fitted only to strictly positive responses. Surprisingly, the volume of the medium also had a significant quadratic term showing that there is an optimal volume of medium in the shake flasks. This supports the idea that a stable standing wave in the flasks is optimal, something that has been noted before (Engel et al. 2011).

5.3 Logistic regression

A logistic regression could be fitted to the data and may be used to predict if pel- letitization will occur under various circumstances. Logistic regression is far more suitable than response surface methodology for modelling pelletization. The logistic regression shows that pelletization depends on pH, solid particles in the medium, agitation and the calcium concentration, in perfect agreement with previous research (Gibbs et al. 2000, Papagianni 2004). The temperature term was also significant. The experimental design by Liu et al. (2008) was very different from the one used in this thesis, making comparisons difficult. The importance of the spore concentration on pelletization can not be questioned, but it was insignificant in our logistic regression. This is because even the high level of inoculum size was much smaller than the critical coagulation concentration.

5.4 Biocapsules

It is possible to immobilize yeast and bacteria in fungal pellets. For this to occur it is important that the medium is selective and only support growth of the filamentous fungus and that the medium is pH-buffered. Because of the lack of a deeper understanding of the mechanisms involved, biocapsules are difficult to achieve but important results have been published in 2011 and the first months of 2012 demonstrating the potential of the technique. The water treatment experiments with biocapsules by Zhang et al. (2011) and the encapsulation of green algae by Zhou et al. (2012) shows that capsules of Aspergillus can contribute to waste water treatment and production of biofuel and biomass using photosynthesis. This thesis have demonstrated that Zygomycetes can grow and forms pellets under the conditions necessary for co-immobilization.

47 Martínez et al. (2011) used a special strain of S. cerevisiae var. capensis, a so-called flor yeast that is used for fermenting sherry wines. These flor yeasts have the unique ability to float to the surface of a culture broth and form a floating waxy biofilm. They are able to do this because of two mutations in the FLO11-gene. There is a deletion in a repression-region in the promoter, so that expression of the FLO11- protein is up-regulated. The second mutation causes the resulting protein to be highly hydrophobic. The resulting flocculin protein is anchored to the outside of the cell membrane with a post-translationally attached GPI-anchor. The cell surface of these flor yeasts hence become much more hydrophobic. This is likely the explanation for why flor yeasts could be immobilized in Penicillium pellets by Martínez et al. (2011). Only the flocculating strain CCUG53310 could be immobilized in our study. This suggests that the physio-chemical mechanism behind the successful encapsulation is hydrophobic interaction. Papagianni (2004, p. 201) anecdotally mentions that factors inducing pelletization of A. niger simultaneously increases the hydrophobicity of the fungal cell wall. van Suijdam et al. (1980, p. 216) writes that spores of A. niger and especially P. chrysogenum are highly hydrophobic and tend to float on the surface in air lift reactors. It can hardly be a coincidence that these hydrophobic fungi are the same that has been shown to spontaneously form biocapsules with hydrophobic yeast and algae. If mutations in the FLO-genes is indeed responsible for the yeast’s ability to be encapsulated, this ability can be transferred to virtually any yeast strain by cloning the mutant FLO11-gene into a vector, something that has already been achieved (Douglas et al. 2007). Most yeast strains are able to flocculate if grown in a medium that induces expression of the FLO-genes. The method developed here, using first a medium to induce pelletization of the filamentous fungus and then a second medium for the growth of the yeast allows for a wide range of physiological conditions that may induce flocculation of yeasts. Primarily, the temperature and pH can be used to induce flocculation. Control of flocculation by manipulation of physiological factors in the medium is however difficult (Zhao & Bai 2009, p. 851). Sucrose is suitable as carbon source in the secondary medium because it is not easily used by the filamentous fungi and it does not interfere with the flocculation of the yeast, regardless of the exact flocculation mechanism. By adapting the secondary medium to the particular yeast strain so that it induces flocculation, our method might be used for encapsulating virtually any yeast that is able to flocculate inside pellets of Zygomycetes.

5.5 Microscopy

Fungal pellets and biocapsules can be embedded in agar or parafﬁn and cut into thin sections allowing for high resolution photography of the interior. If the yeast cells are properly immobilized, they will remain inside the biocapsule during the dehydration procedure. Agar-embedding is useful for temperature-critical samples, but gives poor sections.

48 Parafﬁn is easily cut into sections less than 10 µm. 15–20 µm is the optimal thickness. If the glass slides are treated with gelatin and chromium salt, the sections are easily de-parafﬁnised. Digital image techniques such as focus stacking and photomerging are very useful for microscopy of large specimens.

49 6 Conclusions

In this thesis it has been demonstrated that the morphology of Zygomycetes can be controlled by manipulation of physiological conditions in the growth medium. Sup- plementing YPD-medium with calcium carbonate results in pellets of high quality and high reproducibility. The pelletization depends on many different factors; inoculum size, pH, agitation, solid particles and calcium concentration being the most important. The effect each of these factors have been determined using multiple logistic regression. Pelletization was also achieved in a completely defined medium and in media with xylose as the carbon source. Cells of the flocculating yeast strain CCUG53310 was immobilized inside fungal pellets. Yeast cells can be immobilised in pellets if a co-culture is first grown in a medium inducing pelletization of the filamentous fungus and contains a carbon source not utilized by the yeast, and if the pellets are then moved to a second medium allowing growth and flocculation of the yeast. Several non-flocculating yeast strains could grow inside pellets but they were not immobilized. The successful immobilization of yeast cells inside fungal pellets is dependent on the expression of several genes in the FLO-family. The results in this study sheds new light on the importance of flocculation and its role in immobilization and inhibitor tolerance.

7 Suggestions for future work

The fundamental physical effects causing pelletization remains unknown. Even the coagulation theory is questionable, as it does not really explain the phenomena observed and it does not predict under what circumstances fungi will grow as pellets. A general theory for fungal pelletization is needed. Yeasts with other types of flocculation, such as FLO11-dependent flor-forming yeast can probably be encapsulated in Zygomycetes pellets. It remains to be tested if biocapsules provide protection from inhibitors. Different kinds of flocculating yeasts might be more or less sensitive to inhibitors. It is very laborious and time-consuming to make biocapsules, and they can only be motivated if they enable easy harvest and recycling of biomass for a very long time. The life time and aging of biocapsules should be examined. The logistic regression and response surface obtained might also predict pelletization in other species of Zygomycetes. The models should be validated by new experiments. The expression and regulation of the FLO-genes are very complex. The physiological factors affecting their expression needs to be determined. The structures of the

50 corresponding proteins are not known. In patricular, the different mutant genes of several strains should be sequenced and the protein structures determined. The unknown metabolite ”243” should be identiﬁed, preferably by HPLC-MS.

8 Acknowledgements

The authors would like to thank our many co-workers and express our gratitude over the many helpful discussions, suggestions, critical comments and practical help we have received. Our main supervisor Dr. Patrik Lennartsson has been of utmost value and have helped us on a daily basis. The same is true for PhD-student Johan Westman, who also developed a lot of the microtome techniques. Our examinor Prof. Mohammad Taherzadeh has provided several helpful suggestions. Prof. Kim Bolton and Dr. Peter Ahlström has provided interesting ideas about the physio-chemical basis for the agglomeration of spores. Dr. Elisabeth Feuk-Lagerstedt have made several useful suggestions and participated in valuable discussions. PhD-student Päivi Ylitervo helped in calibrating the pCO2- sensor for the reactor experiments. PhD-student Jorge Ferreira has helped in many ways and in particular by taking samples late at night. Dr. Magnus Lundin was of great help in designing the multifactorial experiment as well as in the interpretation of the resulting data. Research engineer Kristina Laurila has helped a lot with practical matters in the laboratory. Finally we would like to thank PhD-student Thomas Schwartz for critically reading the manuscript.

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57 Appendix A

Medium according to Liao

The Liao medium is based on a medium described by Liao et al. (2007). It is a general medium containing solid particles of calcium carbonate that induces pelleted growth in Rhizomucor and Rhizopus. The glucose may be substituted for xylose (Liaox- medium). 4 g/l potato extract 20 g/l glucose or xylose 6 g/l peptone (from soybeans) 6 g/l calcium carbonate, CaCO3 The sugar is autoclaved separately. A magnetic stirring bar should be included as stirring is necessary to keep the calcium carbonate suspended.

Medium according to Sues

This medium has been used for alcohol production with Zygomycetes, and sometimes using dilute-acid lignocellulosic hydrolysate as carbon source. The medium is optimized for maximal ethanol productivity with Mucor indicus. It has been used by Sues et al. (2005), Karimi et al. (2005) and Karimi et al. (2008). This medium normally does not induce pelleted growth. 30 g/l carbon source (normally glucose, xylose or mixtures of sugars) 5 g/l yeast extract 7.5 g/l ammonium sulphate, (NH4)2SO4 3.5 g/l potassium dihydrogen phosphate, KH2PO4 1.0 g/l calcium chloride dihydrate, CaCl2· 2H2O 0.75 g/l magnesium sulphate heptahydrate, MgSO4· 7H2O Antifoam 1.0 g/l Vitamin solution 1 ml/l Trace metal solution 10 ml/l Pellets can form if zink, vitamins and yeast extract is excluded (Sues et al. 2005).

Medium according to Yang

This medium is from Yang et al. (1995). The glucose may be substituted for xylose to obtain a medium suitable for co-culture with yeast. The medium is deﬁcient in several metal ions, induces physiological stress and growth is very slow. 20 g/l glucose 2 g/l urea 0.6 g/l potassium dihydrogen phosphate, KH2PO4 0.25 g/l magnesium sulphate, MgSO4

58 0.09 g/l zink sulphate, ZnSO4

The medium may be supplemented with 5 ml/l trace metal solution. This reduces stress and increases growth. Sugar, urea and salts should be autoclaved separately and mixed when cool. Trace metals must be sterile ﬁltered and added to cool medium.

Base medium without calcium

The base medium was used in the factorial experiment. 20 g/l xylose 1.80 g/l ammonium chloride, NH4Cl 0.5 g/l magnesium sulfate heptahydrate, MgSO4· 7H2O 1.0 g/l sodium chloride, NaCl 0.6 g/l potassium dihydrogen phosphate, KH2PO4

5 ml/l trace metal solution 40 ml/l pH 5 buffer The xylose was autoclaved separately. Trace metal solution and pH buffer was added to room temperature medium.

Base medium with calcium

The base medium with calcium was identical to the base medium without calcium, but a stock solution of calcium chloride was added at room temperature to a ﬁnal CaCl2 concentration of 5.0 g/l.

Medium according to Xia

Xia et al. (2011) used the following medium for cultivation of Mucor circinelloides and obtained pellets. 20 g/l glucose 1 g/l yeast extract 1.5 g/l ammonium chloride, NH4Cl 6 g/l potassium dihydrogen phosphate, KH2PO4 1.2 g/l magnesium sulphate heptahydrate, MgSO4· 7H2O The medium is adjusted to pH 3.0.

18 hours after inoculation, 4 g/l calcium carbonate, CaCO3 is added causing the pH to increase to 5.3.

59 Medium according to Žnidaršicˇ

This medium was described by Žnidaršicˇ et al. (1998) and used by them for inducing pelletization of Rhizopus nigricans. 20 g/l glucose 6 g/l peptone from soybean 5.7 g/l yeast extract 4 g/l sodium chloride, NaCl 2 g/l dipotassium hydrogen phosphate, K2HPO4 0.5 g/l Tween-80 The medium was adjusted to pH 5.0.

Medium according to Zhou

This medium was used by Zhou et al. (2000) for inducing pelleted growth of Rhizo- pus oryzae. 50 g/l glucose 2 g/l urea 0.6 g/l potassium dihydrogen phosphate, KH2PO4 0.5 g/l magnesium sulphate heptahydrate, MgSO4· 7H2O 0.0176 g/l zink sulphate heptahydrate, ZnSO4· 7H2O 0.498 g/l iron sulphate, FeSO4

Glucose, urea and salts are autoclaved separately.

Soft wood hydrolysate

A dilute sulfuric acid soft wood hydrolysate with pH 1.8 was treated with calcium carbonate until the pH reached 5.5. A precipitate consisting mainly of calcium sulfate was then removed by centrifugation. pH 5 buffer

A pH-buffer for the base medium was done using potassium hydrogen phtalate and sodium hydroxide. Per 100 ml: 50.0 ml 0.100 M KH-phtalate 22.6 ml 0.100 M NaOH And water to a total volume of 100 ml. This buffer has a pH of 5.00 at 20◦C.

60 Trace metals

A trace metal solution described by Taherzadeh et al. (2003, p. 169) was used. The solution contained, per liter:

3.000 g EDTA disodium salt, C10H14N2Na2O8·2H2O 900 mg calcium chloride, CaCl2· 2H2O 900 mg zink sulphate, ZnSO4· 7H2O 600 mg iron(II) sulphate, FeSO4· 7H2O 200 mg boric acid, H3BO3 155.5 mg manganese chloride, MnCl2· 2H2O 80 mg sodium molybdate, Na2MoO4· 2H2O 60 mg cobolt chloride, CoCl2· 2H2O 60 mg copper sulphate, CuSO4· 5H2O 20 mg potassium iodide, KI The salts are dissolved in water. Adjust pH to 4 with NaOH. Add water to a total volume of 1.000 l. Autoclave and store at 4◦C.

Vitamin solution

The vitamin solution has been described earlier by Taherzadeh et al. (2003, p. 169). For 500 ml solution, dissolve 25 mg D-biotin in 10 ml 0.1 M NaOH. Add the dissolved biotin to 300 ml water and adjust pH to 6.5 using 0.1 M HCl. Add the following vitamins: 100 mg p-aminobenzoic acid 500 mg nicotinic acid 500 mg calcium panthotenate 500 mg pyridoxine, HCl 500 mg thiamine, HCl Adjust pH to 0.5 with 2 M NaOH. Add 12.500 g m-inositol and adjust pH to 6.5. The solution is ﬁlter sterilized and stored at 4◦C.

Yeast Nitrogen Base

The secondary medium used for making biocapsules contained a simple inorganic yeast nitrogen base consisting of: 1 g/l KH2PO4 5 g/l (NH4)2SO4 0.5 g/l MgSO4 0.1 g/l NaCl 0.1 g/l CaCl2

61 Lactophenol cotton blue

50 mg methyl blue/aniline blue (not to be confused with methylene blue) 20 g phenol crystals, C6H5OH 20 ml lactic acid 40 ml glycerol 20 ml water First dissolve methyl blue in water over night. On the second day, add phenol to lactic acid and let it dissolve using a magnetic stirrer. Add glycerol to the phenol solution. Mix the two liquids.

62 Appendix B

Design table

(8−2) The multifactorial experiment was done as a 2V fractional central composite design, as described in (Montgomery 2004, p. 331, p. 630). The design contains 64 corner points, 16 face centered axial points and 8 center points. The deﬁning relation is I = ABCDG = ABEFH = CDEFGH. The three respons varables are the number of formed pellets, the average diameter of the pellets, and the ﬁnal pH of the culture. The binary response variable is not displayed.

63 Table B.1: Design table RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH 1 -1 -1 1 -1 -1 -1 -1 1 4 7 4,07 2 -1 -1 -1 -1 1 -1 1 -1 2 5 3,18 3 -1 -1 1 1 -1 1 1 -1 0 5,5 4 -1 -1 -1 1 1 1 -1 1 0 3,09 5 -1 -1 -1 1 -1 -1 -1 1 0 4,23 6 -1 -1 1 -1 1 -1 -1 -1 1 2,93 7 -1 -1 1 1 1 -1 1 -1 136 2,5 3 8 -1 -1 -1 -1 -1 1 1 -1 0 5,94 9 -1 -1 1 -1 1 1 -1 1 0 2,98

64 10 -1 -1 -1 -1 1 1 1 1 0 3,36 11 -1 -1 -1 1 -1 1 -1 -1 0 4,1 12 -1 -1 1 1 -1 -1 1 1 0 5,43 13 -1 -1 -1 1 1 -1 -1 -1 0 2,58 14 -1 -1 1 -1 -1 1 -1 -1 0 3,9 15 -1 -1 -1 -1 -1 -1 1 1 0 5,86 16 -1 -1 1 1 1 1 1 1 50 4 4,05 17 0 0 0 0 0 0 0 0 117 2,5 2,56 18 0 0 0 0 0 0 0 0 281 1,5 2,53 19 -1 1 1 1 1 -1 -1 1 0 3,42 20 -1 1 -1 -1 -1 1 -1 1 0 4,23 21 -1 1 -1 1 -1 -1 1 -1 0 5,91 22 -1 1 1 1 -1 -1 -1 -1 0 3,91 RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH Table B.2: Design table RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH 23 -1 1 1 -1 1 1 1 -1 0 3,87 24 -1 1 -1 -1 -1 -1 -1 -1 0 4,1 25 -1 1 -1 1 1 -1 1 1 0 4,72 26 -1 1 1 -1 1 -1 1 1 0 4,22 27 -1 1 -1 1 -1 1 1 1 0 5,91 28 -1 1 1 -1 -1 1 1 1 0 5,4 29 -1 1 1 1 1 1 -1 -1 0 2,88 30 -1 1 1 -1 -1 -1 1 -1 0 5,44 31 -1 1 -1 -1 1 1 -1 -1 0 2,5

65 32 -1 1 1 1 -1 1 -1 1 0 4,03 33 -1 1 -1 -1 1 -1 -1 1 0 3,26 34 -1 1 -1 1 1 1 1 -1 0 2,69 35 0 0 0 0 0 0 0 0 199 1,5 2,74 36 0 0 0 0 0 0 0 0 140 1,5 3,36 37 1 -1 -1 1 -1 -1 1 -1 3 6 4,51 38 1 -1 1 -1 1 1 1 -1 21 6 2,78 39 1 -1 -1 -1 1 -1 -1 1 28 10 2,81 40 1 -1 1 1 -1 -1 -1 -1 1 3 4,83 41 1 -1 1 1 1 -1 -1 1 6 14 3,56 42 1 -1 -1 -1 -1 1 -1 1 0 2,71 43 1 -1 -1 1 1 -1 1 1 0 2,68 44 1 -1 1 -1 -1 -1 1 -1 6 4,88 RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH Table B.3: Design table RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH 45 1 -1 -1 1 1 1 1 -1 0 2,46 46 1 -1 1 -1 -1 1 1 1 0 3,88 47 1 -1 1 1 -1 1 -1 1 0 4,78 48 1 -1 -1 -1 -1 -1 -1 -1 4 6 3,93 49 1 -1 -1 -1 1 1 -1 -1 0 2,49 50 1 -1 1 -1 1 -1 1 1 2 14 4,36 51 1 -1 -1 1 -1 1 1 1 3 5 5,11 52 1 -1 1 1 1 1 -1 -1 15 6 2,79 53 0 0 0 0 0 0 0 0 127 2,5 2,63

66 54 0 0 0 0 0 0 0 0 380 3,5 2,61 55 1 1 -1 -1 -1 -1 1 1 0 2,32 56 1 1 1 -1 -1 -1 -1 1 20 1 4,77 57 1 1 -1 -1 1 -1 1 -1 0 2,36 58 1 1 1 1 -1 1 1 -1 0 5,23 59 1 1 -1 1 -1 -1 -1 1 0 2,52 60 1 1 1 -1 1 -1 -1 -1 170 3 2,5 61 1 1 1 1 -1 -1 1 1 40 4 4,28 62 1 1 -1 1 1 -1 -1 -1 0 2,46 63 1 1 1 -1 -1 1 -1 -1 0 3,32 64 1 1 -1 -1 1 1 1 1 0 2,33 65 1 1 -1 1 -1 1 -1 -1 0 3,03 66 1 1 1 1 1 -1 1 -1 0 2,21 RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH Table B.4: Design table RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH 67 1 1 -1 -1 -1 1 1 -1 0 4,35 68 1 1 1 -1 1 1 -1 1 0 2,29 69 1 1 -1 1 1 1 -1 1 0 2,37 70 1 1 1 1 1 1 1 1 115 5 2,5 71 0 0 0 0 0 0 0 0 138 2 2,56 72 0 0 0 0 0 0 0 0 183 2 2,62 73 -1 0 0 0 0 0 0 0 152 2 4,14 74 1 0 0 0 0 0 0 0 40 4,47 75 0 -1 0 0 0 0 0 0 90 4 2,26

67 76 0 1 0 0 0 0 0 0 198 2 2,57 77 0 0 -1 0 0 0 0 0 25 3,5 3,08 78 0 0 1 0 0 0 0 0 4 5 4,89 79 0 0 0 -1 0 0 0 0 25 1,5 4,3 80 0 0 0 1 0 0 0 0 14 1,5 4,93 81 0 0 0 0 -1 0 0 0 0 4,99 82 0 0 0 0 1 0 0 0 60 3,5 2,72 83 0 0 0 0 0 -1 0 0 2 6 3,54 84 0 0 0 0 0 1 0 0 6 3 4,67 85 0 0 0 0 0 0 -1 0 86 0 0 0 0 0 0 1 0 0 5,52 87 0 0 0 0 0 0 0 -1 4 1,5 4,54 88 0 0 0 0 0 0 0 1 3 1 4,81 RunOrder Temp Agitation Calcium Surfactant Spore Particles pH Volume Pellets Diameter Final pH This page intentionally left empty.