Edible Fungal Production using Acetic Acid as Carbon and Energy Source

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DIBLE

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UNGAL

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RODUCTION USING

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CETIC

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CID AS

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ARBON AND

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NERGY

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OURCE

BSc in Chemical Engineering Applied Biotechnology Anna Axebrink Barbara Mae Alontaga

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Programme: Chemical Engineering – Applied Biotechnology

Swedish title: Produktion av ätbara svampar med ättiksyra som kol och energikälla English title: Edible Fungal Production using Acetic Acid as Carbon and Energy Source Year of publication: 2020

Authors: Anna Axebrink, Barbara Mae Alontaga

Supervisor: Rachma Wikandari,S. TP., M. Biotech., Ph.D., Dr. Ria Millati, S.T., M.T. Examiner: Assoc. Prof. Ilona Sárvári Horváth

Key words: VFA, Volatile fatty acid, acetic acid, Fungi, Carbon source, Energy source __________________________________________________________________________

Abstract

Volatile fatty acids (VFAs) have become attractive and gained high research interest due to its significance for the chemical industry and economical advantage. These acids can be produced by utilizing organic waste such as food waste as substrate through anaerobic digestion. Anaerobic digestion is an environmental process that occurs naturally and produces biogas as the main product. VFAs are intermediate products formed during anaerobic digestion where acetic acid, a type of VFA, is the primary product. The main objective of this study was to utilize acetic acid as carbon and energy source for production of edible fungi, Rhizopus ologisporus, Mucor indicus and Volvariella volvacea.

The first step was to evaluate if acetic acid could be used as carbon and energy source for edible fungi production. The results showed, that acetic acid is suitable as carbon and energy source for fungal biomass production. The second step was to optimize growth in liquid media. The cultivations were carried out by using five different conditions, where the liquid media contained different combinations of acetic acid, yeast extract and minerals as well as comparing orbital and linear oscillations. _{Fungal cultivation was possible regardless of the}

medium composition and type of water shaking baths. However, a linear water shaking bath with a combination of acetic acid yeast extract and/or minerals seems to be the best.

Finally, as step three, acetic acid concentrations, 0.2 g/l and 2.0 g/l were used under similar conditions as in step two to see whether a higher concentration of acetic acid would be beneficial. Although the cultivation containing 2.0 g/l gave a higher value of dry weight, the value of yield is questionable. Further studies are needed to confirm if a higher concentration is beneficial or if it might act as an inhibitor for fungal cultivation.

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Sammanfattning

Flyktiga fettsyror (VFAs) har ekonomiska fördelar och kan användas inom kemiska industrier i olika sammanhang, detta har lett till ett stort forskningsintresse för att kunna nyttja VFAs. Organiskt avfall, såsom matavfall, kan användas som substrat för att producera fettsyror genom anaerob rötning. Anaerob rötning är en miljövänlig process och VFAs bildas som intermediära produkter under den anaeroba nedbrytningen där annars bildas biogas som slutprodukt. Syftet med denna studie var att använda ättiksyra, (den vanligaste typen av VFAs), som kol- och energikälla vid odling av tre olika ätbara svampar, som Rhizopus oligosporus, Mucor indicus, och Volvariella volvacea.

Först odlades dessa ätbara svampar i odlingsmedium innehållande ättiksyra. Resultatet visade

att ättiksyra kan användas som kol- och energikälla vid produktion av svampbiomassa. Målet

i de nästkommande stegen var att optimera tillväxtförhållande för svampodlingen. Fem olika odlingsmedier som innehöll olika kombinationer av ättiksyra, jästextrakt och mineraler användes. Det undersöktes dessutom hur två olika skakmetoder, orbitalt, eller linjärt, skakbad påverkar odlingen. Svamptillväxt var möjligt vid alla olika förhållanden oavsett sammansättningen av medium och typ av skakbad, däremot verkar odlingsmedium som innehåller ättiksyra, jästextrakt och/eller mineraler i kombination med linjär skakning vara de bästa förutsättningar för tillväxt av biomassa. I det sista steget kultiverades svamp med olika koncentrationer av ättiksyra, 0,2 g/l och 2,0 g/l, under liknande optimerade förhållanden som ovan, för att undersöka om en högre koncentration av ättiksyra skulle vara fördelaktig. Det producerades mer svampbiomassa (som torrvikt) vid koncentration av 2,0 g/l ättiksyra jämfört med när 0,2 g/l ättiksyra användes, dock var det svårt att säkerställa utbytet. Det behövs därför ytterligare fortsatta studier för att kunna bevisa om en högre koncentration av ättiksyra är fördelaktig för odlingen, eller om en högre koncentration skulle verka inhiberande för tillväxten.

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1. INTRODUCTION

1.1 Background

Volatile fatty acids, VFAs can be produced easily at a low cost from all biomasses and are very sustainable and extremely useful. Nowadays, VFAs are used in different applications such as bioplastic or polyhydroxyalkanoates (PHAs) production, biohydrogen and biogas production, nutrient removal in biological wastewater treatment, in the chemical industry (Strazzera et al., 2018) or for lipid production (Huang et al., 2016). With the increasing interest of utilizing organic waste, organic degradation processes could be a source of recovering VFAs which in turn could be used as substrate, thus creating a more sustainable source of carbon (Zacharof and Lovitt, 2014). As approximately one third of worldwide produced food is wasted, this combined with heightened environmental awareness, has according to Jin et al. (2002) led to finding a variety of ways to utilize food waste as a substrate for producing e.g. food protein. Another alternative, the production of VFAs from food waste through anaerobic digestion (Greses et al., 2020).

Food waste has a high content of moisture and organic matter, therefore it is an ideal substrate for anaerobic digestion. It would be advantageous to utilize food waste to produce VFAs instead of disposing food waste to sanitary landfill, which can cause environmental and health problems such as emission of greenhouse gases and limited landfill space. Anaerobic digestion is an eco-friendly bioprocess that consists of four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis. As VFAs are produced in the first three steps, the methanogenesis process should be avoided before the substrate (food waste) is converted into biogas (Jiang et al., 2013). When producing VFAs, acetic acid is produced as a principal product (Cheah et al., 2019). For a better yield of acetic acid during production of VFAs, a higher pH and temperature is beneficial (Garcia-Aguirre et al., 2017).

Acetic acid is used in various products and is industrially a very important organic chemical (Huo et al., 2015). It comprises of two carbon atoms. Theoretically, it can be metabolized by organisms via a single step conversion into acetyl-CoA by acetyl-CoA synthase (Bhatia et al., 2019). Therefore, it could be a good substrate for cultivation a valuable biomass such as edible fungi or mushroom.

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Fungal or mushroom cultivation becomes popular due to high economic value of biomass as well as health benefits. There are many identified species of fungi, which are known for a significant global contribution to human food and medicine. Useful fungi defined as having edible and medicinal value (Oyedele et al., 2018). Edible fungi are nutritious food that can be consumed by humans and other living organisms without causing any side effects. There is evidence for that consuming fungi as a source of high quality protein source can lead to lowering cholesterol (Valverde et al., 2015) as well as have positive impact on the level of insulin in the blood (Vitak et al., 2017). Among the edible fungi/mushroom species, there are several edible fungi and mushrooms that are commonly studied/consumed including R. oligosporus, M. indicus and V. volvacea. M. indicus is used in food and beverages production, whereas R. oligosporus is traditionally used to make tempeh in Indonesia. V. Volvacea, is a mushroom, that belongs to vegetables category, which is valued as it is rich in protein, have unsaturated fatty acids and high content of vitamins (Oyedele et al., 2018). Tempeh and mushroom production in Indonesia gives positive impact not only to the farmers but also to Indonesian people due to simple and low-cost production as well as it is very affordable for consumers (Febrianda and Tokuda, 2017).

Although production of edible fungi and mushroom from volatile fatty acids particularly acetic acid is plausible, however, there is a limited information reported about it. Former students, studying at Universitas Gadjah Mada, have written reports about cultivating fungi with VFA as carbon source. One undergraduate thesis report, written by Fanani (2019), concludes that future studies are highly encouraged as fungal biomass produced from VFA has a potential as animal feed or act as an active compound in food as well as has potential in the nutritional industry. The author cultivated R. oligosporus with a concentration of VFA at 2 g/l and gained a yield of 49 mg/g when cultivating in Erlenmeyer flasks.

Utilization of VFAs derived from agro-industrial residue or food waste not only beneficial from environmental perspective, but also could lowering the production cost (Souza Filho et al., 2019). As acetic acid is one of the major products when producing VFAs (Yuan et al., 2011), therefore, researching whether it alone could be utilized as a carbon and energy source for growing fungal biomass as well as whether it is possible to use VFA containing cultivation medium for biomass production of edible fungi and mushroom is of interest.

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1.2 Aim and objective

The general objective of the study was:

• To investigate the potential for VFA, as cultivation medium for the production of edible fungi and mushrooms

The specific objectives of the study were:

• To find out the most suitable medium for spore production • To investigate the potential of acetic acid as a sole carbon source

• To investigate the effect of addition of yeast extract and minerals on biomass production

• To investigate the effect of acetic acid concentration on biomass production

1.3 Research questions

Some of the questions that emerged when working with VFA were: • Can acetic acid be used as a carbon source?

• Can yeast extract act as carbon source for growth? • Is yeast extract or minerals necessary for growth? • Is a higher concentration of acetic acid beneficial?

This study will not go into depth to answer all these questions, this study will however, act as a starting point of cultivating fungi with acetic acid as carbon source.

2. LITTERATURE STUDY

2.1 Edible fungi

Since ancient times, edible mushroom has been an important ingredient for a nutritional diet. All over the world cultivation of fungi, such as Pleurotus ostreatus, has increased tremendously over the last decades, this might be as it is a nutritional balanced type of food (Chae and Ahn, 2013). As growing mushrooms can be very profitable as seen in the Kilimanjaro region of Tanzania, where farmers have been able to save money in order to pay for their children’s school tuitions as well as expanding and establishing an additional source of income (Muhanji, 2010), there are still some raw materials and preparations of selective compost that drives the costs up. New methods for utilizing e.g. food waste is essential, not only to reduce cost but also managing disposal of organic waste (Chae and Ahn, 2013).

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Depending on the type of mushroom, different substrates are needed in order to produce fruiting bodies. Lentinula edodes, commonly known as Shiitake, requires hard wood saw dust in order to reach a preferable yield (Nitta et al., 2016), whereas P. ostreatus can be grown on rice straws, wheat, ragi, maize, cotton stalk and popular saw dust, to mention some of the possible substrates (Das and Mukherjee, 2007).

The worldwide commercial cultivation of mushrooms is important, some aspects to why cultivation of mushrooms is so widespread might be because of pleasant taste, high quality protein content as well as the ability to grow with low investments and simple techniques. Another contributing fact for some types of mushrooms might be the easy access of raw materials as well as the ability to use waste from agribusinesses (Colavolpe and Albertó, 2014).

According to Colavolpe and Albertó (2014), several countries have an abundance of agro-industrial wastes. One example of a suitable substrate for producing edible mushrooms is sawdust from eucalyptus and populus trees. By using the abundance of sawdust obtained from e.g. production of furniture, regional economies of the country might be improved (Colavolpe and Albertó 2014).

In this study there were three different types of fungi investigated, all can be found in Indonesia, Yogyakarta.

2.1.1 Rhizopus oligosporus

R. oligosporus is a food safe filamentous fungus (Figure 1), mainly used through fermentation of soybeans to produce a traditional Indonesian food called Tempeh (Feng et al., 2007). When making soybean tempeh, R. oligosporus binds the soybeans together with its white mycelium, creating a protein rich cake. The fungus releases enzymes that makes the product easier to digest (Jennessen et al., 2008b). R. oligosporus grows better in high humidity levels, between 75 and 78 %, and an ideal temperature for the growth is 37 °C (Babu et al., 2009).

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Figure 1. Mycelia and spores of R. oligosporus under microscope magnified 40x 2.1.1.1 Taxonomy

Taxonomy of Rhizopus oligosporus (Cantabrana et al., 2015). Kingdom: Fungi Division: Zygomycota Class: Zygomycetes Order: Mucorales Family: Mucoraceae Genus: Rhizopus

Species: Rhizopus oligosporus 2.1.2 Mucor indicus

M. indicus, is a non-pathogenic dimorphic microorganism (Figure 2), which can grow either as yeast or mycelium (Lennartsson et al., 2009). It is one of the most widely used strains of zygomycetes fungi. This fungus is commonly used for production of several foods and beers. M. indicus can assimilate several sugars and grow on different types of substrates such as lignocellulosic hydrolysates which are mixtures of pentoses, hexoses and different fermentation inhibitors, (Karimi and Zamani, 2013) it can also produce a high yield of ethanol (Sues et al., 2005). Besides from being used for the previously mentioned products, the biomass of this fungus can also be used as a rich nutritional source, e.g. fish feed (Karimi and Zamani, 2013).

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Figure 2. Mycelia and spores of M. indicus under microscope magnified 40x 2.1.2.1 Taxonomy

Taxonomy of Mucor indicus (Walther et al., 2019). Kingdom: Fungi Division: Zygomycota Class: Zygomycetes Order: Mucorales Family: Mucoraceae Genus: Mucor

Species: Mucor indicus 2.1.3 Volvariella volvacea

V. volvacea is an edible straw mushroom (Figure 3) that is widely cultivated in Southeast Asia due to its use as a high-quality nutritious food source. Substrates that are used for cultivation of V. volvacea are agricultural wastes such as cotton waste and rice waste (Bao et al., 2013). The straw mushroom produce fruiting bodies which is at first egg shaped, then becomes bell shaped with a characteristic volva and has a greyish-brown cap (Mau et al., 1997). According to Bao and Wang (2016), V. volvacea is one of the most important cultivated edible mushrooms worldwide and currently ranks fifth among cultivated species (Cai et al., 1999).

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10 Figure 3. Mycelia of V. volvacea under microscope magnified 40x 2.1.3.1 Taxonomy

Taxonomy of Volvariella volvacea (Dutta et al., 1936). Kingdom: Fungi Division: Basidiomycota Class: Basidiomycetes Order: Agaricales Family: Pluteaceae Genus: Volvariella

Species: Volvariella volvacea

2.2 Volatile fatty acids

Volatile fatty acids (VFAs) are short-chained acids comprised of two to six carbon atoms in the molecule. Acetic, propionic, butyric, isobutyric, valeric, isovaleric and caproic acid are examples for the different variety of VFAs (Lee et al., 2014). These acids have gained high research interest due to their importance for the chemical industry as well as their low production cost. VFAs are essential intermediate products formed when organic materials are broken down by anaerobic digestion (Lukitawesa et al., 2020). As research interest increases, applications of VFAs become wider. Nowadays, these fatty acids are utilized in various applications. One of these applications is for fungi cultivation where these acids are utilized as carbon source (Bhatia et al., 2019).

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11 2.2.1 Types of VFA

Acetic acid (C2H4O2 or CH3COOH)

Acetic acid is a weak acid that consists of two carbon atoms (Figure 4). It has antibacterial and antifungal properties and is found most commonly in vinegar. This is an essential acid and is used in various applications. Acetic acid is a colorless liquid with a sharp, distinctive odor and is commonly used in the food industry as an acidity regulator (National Center for Biotechnology Information).

a) b)

Figure 4. a) 2D structure and b) 3D structure of acetic acid (National Center for Biotechnology Information) Propionic acid (C3H6O2 or CH3CH2COOH)

Propionic acid is a short-chain saturated fatty acid containing ethane in the carbon chain (Figure 5). This acid is colorless and has a sharp rank odor, which produces irritating vapor. Propionic acid is widely used as an antifungal drug as well as food preservative in food industry. In addition, the acid can be found as an end product in humans and other mammals when digesting carbohydrates (National Center for Biotechnology Information).

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12 Butyric acid (C4H8O2 or CH3CH2CH2COOH)

Butyric acid is a four-carbon saturated fatty acid (Figure 6) and appears as a colorless liquid. It has a penetrating and unpleasant odor with acrid taste. This acid is commonly found in ester form in animal fats and plant oils. Butyric acid with low-molecular weight esters have pleasant aromas or tastes, therefore, they are used as food and perfume additives (National Center for Biotechnology Information).

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Figure 6. a) 2D structure and b) 3D structure of butyric acid (National Center for Biotechnology Information) Isobutyric acid (C4H8O2 or (CH3)2CHCOOH)

Isobutyric acid is a carboxylic and a branched-chain saturated fatty acid consisting propionic acid with a methyl branch at C-2 (Figure 7). This acid appears as a colorless liquid with a light odor of rancid butter. It can be produced via microbial (gut) metabolism or can be found in certain foods and fermented beverages. Isobutyric acid can irritate the nose, throat and lungs if breathed in and can burn the skin and eyes if absorbed. The acid is very soluble in ethanol, ether and organic solvents but less soluble in water (National Center for Biotechnology Information).

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Figure 7. a) 2D structure and b) 3D structure of isobutyric acid (National Center for Biotechnology Information) Valeric acid (C5H10O2 or CH3(CH2)3COOH)

Valeric acid, also known as pentanoic acid, is a straight-chain saturated fatty acid comprised of five carbon atoms (Figure 8). It has a very unpleasant odor and can be found naturally in a perennial flowering plant, valerian. This acid has a function as a plant metabolite, also its primary use the synthesis of its esters. The volatile esters of valeric acid have pleasant odors

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and are utilized in perfumes and cosmetics, also some have fruity flavors and are used as food additives (National Center for Biotechnology Information).

a) b)

Figure 8. a) 2D structure and b) 3D structure of valeric acid (National Center for Biotechnology Information) Isovaleric acid (C5H10O2)

Isovaleric acid is a C5, natural, branched-chain saturated fatty acid (Figure 9) that can be found in plants and essential oils. This acid is a colorless liquid and is well soluble in most common organic solvents but sparingly soluble in water. Isovaleric acid has a function as a mammalian metabolite and a plant metabolite (National Center for Biotechnology Information).

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Figure 9. a) 2D structure and b) 3D structure of isovaleric acid (National Center for Biotechnology Information) Caproic acid (C6H12O2 or CH3(CH2)4COOH)

Caproic acid, also known as hexanoic acid, is a straight-chain saturated fatty acid consisting of six carbons (Figure 10). This acid can be found naturally in plants, animal fats and oils. Caproic acid has a function as a plant metabolite and a human metabolite (National Center for Biotechnology Information).

a) b)

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2.2.2 Concentration of VFAs derived from organic waste

Food waste is an organic waste with a high content of moisture and organic matter, therefore it is an ideal substrate for anaerobic digestion. Anaerobic digestion is an eco-friendly bioprocess used to treat organic wastes and consists of four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis. VFAs are produced in the first three steps, therefore, if the aim is VFA production, the last step, methanogenesis, has to be inhibited (Jiang et al., 2013). According to Lukitawesa et al. (2020), the concentration and type of VFAs, produced through this modified anaerobic digestion process, depend on the composition of the substrate, operational parameters, available microbial community, type of reactor and process design.

Figure 11. Metabolic pathways for organic acids production (Feng et al., 2009).

The production of VFAs from organic waste through anaerobic digestion begins from hydrolysis where complex organic matter (carbohydrates, proteins and lipids) in waste are broken down into simpler organic monomers, such as sugars, amino acids and long chain fatty acids (LCFA) by enzymes. Acidification (acidogenesis and acetogenesis) occur when the hydrolysed organic monomers are converted into mainly VFAs, such as acetic, propionic and butyric acids (Lukitawesa et al., 2020, Batstone et al., 2002).

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As seen in Figure 11, metabolic pathways for organic acids production, the enzymes phosphotransacetylase (PTA) and phosphotransbutyrylase (PTB) play a key role in the production of acetic and butyric acid. These enzymes convert acetyl-CoA and butyryl-CoA into acetyl and butyryl phosphate. Acetyl phosphate is then converted into acetic acid by acetate kinase (AK) and by butyrate kinase (BK) the butyryl phosphate is converted into butyric acid. In a different way, pyruvic acid is converted into oxaloacetic acid and methylmalonyl-CoA is converted into propionyl-CoA by oxaloacetate transcarboxylase (OAATC). Finally, propionyl-CoA is converted into propionic acid by the action of CoA-transferase (Feng et al., 2009).

2.2.3 Acetic acid

For this study, acetic acid will be used as carbon and energy source. Acetic acid is used in various products and is industrially a very important organic chemical (Huo et al., 2015). It comprises of two carbon atoms. Utilizing acetic acid as carbon source for fungi cultivation is beneficial since it can be easily consumed compared to other VFAs. One possible reason for easier consumption of acetic acid might be that, its conjugate base acetate can be directly utilized in a single step conversion into acetyl-CoA by acetyl-CoA synthase, whereas other VFAs require extra steps to be metabolized (Bhatia et al., 2019). Additionally, when producing VFAs through anaerobic digestion, acetic acid is the major product (Cheah et al., 2019) due to its flexibility as it can be produced through both acidogenesis and acetogenesis processes. In acidogenesis process simple sugars are broken down into acetic acid while in acetogenesis process, longer VFAs, comprising from three carbons or more, are oxidized into acetic acid (McPhail et al., 2012). For a better yield of acetic acid during production of VFAs, a higher pH and temperature is beneficial (Garcia-Aguirre et al., 2017).

2.3 Factors influencing aerobic submerged cultivation

Submerged cultivation, also known as fermentation, is a metabolic process induced by microorganisms that converts organic substrates into useful products. It is one of the oldest methods of food preservation. This process can be classified as solid-state fermentation and submerged fermentation. Both fermentation processes are successful and productive depending on their applications.

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occurs when oxygen is introduced into the fermentation vessel, whereas anaerobic fermentation, occurs when the presence of oxygen is little or absent. Aerobic fermentation is a faster and more intense process than anaerobic fermentation. Also, the biomass produced from aerobic fermentation has high cell density due to high agitation and aeration rate (Ojewumi, 2018).

2.3.1 Medium composition

The composition of the medium is an essential during a cultivation process. Media for cultivation should be comprised of water, sources of energy, carbon, nitrogen and other minerals.

Water is the major component of all cultivation media. It is important and is needed when heating, cooling, cleaning and rinsing. Clean water is required and essential to consider pH, to dissolve salts and to prevent contaminations. The source of energy for microbial growth comes from chemical oxidation reactions or from converting light energy, whereas the source of carbon is commonly coming from carbohydrates, lipids and proteins, or from CO2. Also,

some microorganisms have potentials to utilize methanol or hydrocarbons as carbon and energy sources.

Nitrogen can either be taken from organic or inorganic sources. Organic nitrogen sources are supplied as amino acids, proteins or urea, whereas inorganic nitrogen sources are supplied as ammonia gas, ammonium salts or nitrates. Other minerals are also required for the growth and metabolism of microorganisms. Magnesium, phosphorus, potassium, sulphur, calcium and chlorine are examples for essential mineral sources (Stanbury et al., 2013).

The media used in this study, for the cultivation and sporulation of fungi, were potato dextrose agar (PDA) (Merck, Germany) and malt extract agar (MEA) (Merck, Germany). Both were chosen as previous studies showed good experiences with using these selected media. In a book written by Crueger et al. (1990) it is stated that for cultivating many types of fungi, maltose extract is an excellent substrate, and in the text Production of edible fungi from potato protein liquor (PPL) in airlift bioreactor written by Souza Filho et al. (2017) potato dextrose was successfully used as cultivation medium.

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17 2.3.2 pH

One of the parameters that is needed to be continuously monitored to achieve the maximum efficiency of cultivation is pH. All microorganisms have a definite pH range for optimal growth, and it is necessary to maintain the stability of pH throughout the process. pH can harm microorganisms by inhibiting enzyme activity or disrupting the plasma membrane when severe variations in environmental pH occur. During a cultivation processes, initial pH is needed to be measured, and sample withdrawals can be necessary for pH determination throughout the process.

The changes of pH are caused by the design and composition of the medium or by the activity of microorganisms. When microorganisms grow, they release metabolites into the medium that can change the pH, therefore it is preferable if the pH can be continuously monitored and maintained at optimum level (Mulay and Khale, 2011). The pH can be adjusted by the addition of appropriate amount of for example hydrochloric acid (HCl) or sodium hydroxide (NaOH) (Lennartsson et al., 2012, Richter et al., 2014).

2.3.3 Agitation and aeration

Agitation is essential to ensure optimum supply of nutrients in the culture as well as to prevent accumulation of toxic metabolite byproducts. Proper and good mixing generates favorable and homogenous growth environment as well as good product formation. However, excessive mixing can damage the microbial cells and may increase the temperature of the medium as well as may cause increased foam formation. For cultivation in bioreactor, agitation can easily form foam since the media used are often rich in proteins. Therefore, to reduce and avoid foam formation, antifoam agents should be added (Mulay and Khale, 2011).

Aeration is required for successful functioning of aerobic fermentation. It is extremely important to supply sufficient amount of dissolved oxygen to the microorganisms to meet their needs at any stage in the process (Brierley and Steel, 1959). It is necessary to monitor and maintain the conditions of agitation and aeration to minimize foam formation. Foaming is undesirable. It creates disadvantages in cultivation such as lower mass and heat transfers rates as well as invalid process data due to interference at the electrodes (Atri and Ashrafizadeh, 2010).

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18 2.3.4 Acid concentration

From previous studies such as Utilization of food waste-derived volatile fatty acids for production of Rhizopus oligosporus biomass using airlift bioreactor written by Fanani (2019), acetic acid was determined to have a concentration of maximum 2.0 g/l, therefore, acetic acid was primarily investigated in this study as a potential carbon and energy source for the cultivation of edible fungi.

3 MATERIAL AND METHODS

3.1 Material

3.1.1 Fungal strains

The fungal strains used in this study were strains of R. oligosporus, V. volvacea and M. indicus. The fungi were obtained from Biotechnology Laboratory, Department of Food and Agricultural Product Technology, UGM, whereas the mushroom was obtained from the mushroom plantation CV Volva located at Sleman, Yogyakarta, Indonesia. The strains were chosen due to their generally rapid growth as well as easily accessible knowledge from previous laboratory experiments in the field.

3.2 Method

All laboratory work was conducted at the laboratory at UGM, Yogyakarta, and under sterile conditions. By working under sterile conditions, working benches as well as the laborants hands were disinfected with 70% ethanol. All sterile work was done using a burning flame on site to minimize risk of contamination.

3.2.1 Preliminary study

The preliminary study was conducted in order to assess which of the two media was better suited for maintaining the strains for further studies. In addition, a standard curve was determined, where the correlation of absorbance and the number of spores was followed up for the two filamentous fungi investigated. It was later be used in other laboratory works as a tool for calculating number of spores in a solution.

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3.2.1.1 Maintenance on agar plates and determining the standard curve Fungal strains

Edible filamentous fungi R. oligosporus and M. indicus were used.

Agar media

Two different types of media, one containing 39 g/l potato dextrose agar (Merck, Germany) powder and the second containing 48 g/l malt extract agar (Merck, Germany) powder were used for comparison.

Malt extract agar (MEA) and potato dextrose agar (PDA) media were prepared by dissolving each type of agar powder in distilled water. The mixtures were then heated until the powder dissolved thus making the mixtures become clearer (Figure 12).

a) b)

Figure 12. Comparison of mixture a) before and b) after heating

All agar media were transferred in separate Erlenmeyer flasks and sterilized in an autoclave at 121 °C for 1 hour. In addition, petri dishes were dry sterilized at 155 °C for 3 hours.

Casting agar plates

When cooled down until approximately 54 °C, agar media were poured into sterile petri dishes, with approximately 15 ml of agar medium for each plate. Then, when the agar medium had solidified, i.e. approximately 20 minutes later, the petri dishes were flipped and put in an incubator at 37 °C for three days. The agar plates were controlled daily to detect any contamination before inoculating them with fungal spores.

Inoculation

The prepared MEA and PDA plates were inoculated with R. oligosporus and Mucor indicus. Inoculation was made under sterile conditions with inoculating loop.

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Plates containing Mucor indicus were incubated at 28 °C for 7 days whilst plates containing R. oligosporus were incubated at 37 °C for 4 days. Daily controls and visual comparison of sporulation on MEA and PDA, as cultivation media, were conducted.

Spore suspension

Methods to prepare, cultivate and thus obtain a standard curve were similarly done as described above. For this part, only PDA was used as cultivating media and 0.1 g/l chloramphenicol (Sigma, Germany) dissolved in 96 % ethanol was added in order to reduce risk of contamination. Inoculation of fungi was carried out as soon as the agar had solidified. M. indicus were incubated at 28 °C for 7 days and R. oligosporus were incubated at 37 °C for 4 days.

All equipment needed for spore suspension such as micropipette-tips and tubes filled with 5 ml and 10 ml distilled water were autoclaved (1 hour at 121 °C) together with glass cell spreaders and an Erlenmeyer flask containing 180 ml distilled water.

For spore suspension, the cultivated PDA plates containing fungal spores were flooded with 10 ml distilled water. Spores were suspended by gently stroking the PDA plates with a glass cell spreader. The collected spores were then diluted three times with distilled water and a spectrophotometer set to a wavelength of 660 nm was used for determining the absorbance for each diluted spore solution. In addition, the number of spores in the original spore suspension was counted by using a haemocytometer. The concentration of the spore solutions was calculated and a standard curve for each fungus was created. Contaminated plates were utilized as study material with microscope for morphology observations.

3.2.2 Fungal cultivation using acetic acid as main carbon source Fungal strains

Edible filamentous fungus R. oligosporus and edible mushroom V. volvacea were used in these experiments.

Casting agar plates

All agar plates were casted with the same method as for preparing spore suspension. Fungal spores of R. oligosporus where grown under the same conditions and later suspended as described above.

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For V. Volvacea, casting agar plates was also done as described for preparing the spore suspension. However, here the plates were later inoculated with mycelia of V. volvacea and incubated at 28 °C for 7 days.

Preparing liquid cultivation

Two types of liquid media solutions (Table 1 and 2) were prepared in 250 ml Erlenmeyer flasks. The prepared media were then autoclaved for 1 hour at 121 °C. Due to the risk of decomposition of vitamins and other complex nutrients, reactions between sugars and amino acids as well as important pH changes and formation of toxic compounds during sterilization (van Bragt et al., 1971), yeast extract, acetic acid and the minerals listed in Table 2 were autoclaved separately.

Inoculation

Approximately 36 mm2_{was cut out from a petri dish containing mycelia of V. volvacea and}

used as inoculum. For inoculation with R. oligosporus, 1 ml spore suspension with a concentration of approximately 1,8 x 104 _{spores per ml was used.}

The inoculated Erlenmeyer flasks were left for 7 days in an orbital water shaking bath at 30°C with 114 rpm. Samples for checking pH and the level of acetic acid were taken in every 24 hours. The pH was controlled by using pH indicator strips, all adjustments of pH was made by adding 2 M NaOH or 1 M HCl (Richter et al., 2014, Komáromy et al., 2019, Lennartsson et al., 2012). Samples of 1.5 ml taken from the Erlenmeyer flasks of each cultivation were placed in Eppendorf tubes and then analyzed for the level of acetic acid. Centrifugation of the Eppendorf tubes (at 12 000 rpm for 10 mins) was necessary before analysis in order to remove any unwanted substances, such as fungal biomass. Then approximately 1 ml supernatant was taken and used for the analysis by gas chromatography.

Dry fungal biomass, from all cultivations, was obtained by filtering the growth media with pre-dried and weighed filter papers. The filtered biomass was dried in the oven at 105 °C for 24 hours. The final dry weight of the produced biomass as well as the calculated consumed acetic acid were used in calculating yields. Concentrations of acetic acid were used to create a curve showing the consumption of acetic acid over time for each medium investigated. All experiments and analysis of samples were carried out in duplicates. To differentiate the flasks with the same media, the flasks were numbered.

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Table 1. Composition of liquid medium 1

Chemical Amount

Distilled water 100 ml

Acetic acid (Merck, Germany) 0.2 g/l

Acetic acid (Merck, Germany). 0.2 g/l Yeast extract (OXOID, England) 1 g/l MgSO4 * 7H2O (BDH Laboratory supplies,

England)

0.75 g/l

KH2PO4 (Merck, Germany) 3.5 g/l

CaCl2 * 2H2O (Merck, Germany) 1 g/l

(NH4)2SO4 (Merck, Germany) 7.5 g/l

3.2.3 Optimization of growth in liquid medium using different media compositions Preparations for inoculation were conducted the same as in 3.2.2.

In order to determine where the fungi utilise carbon for growth, three types of liquid media solutions were prepared, as shown in Table 3, 4 and 5. Cultivations were carried out in 250 ml Erlenmeyer flasks with 100 ml as working volume. All flasks were inoculated with R. oligosporus. In addition to finding the carbon source, a small test of comparing linear and orbital shaking was made to see whether the fungal growth was affected.

Erlenmeyer flasks were left for 7 days in an orbital shaking water bath at 30°C with 114 rpm. The flasks made for comparing effect of linear shaking instead of orbital shaking, were put in a water bath at 30 °C with 118 oscillations min1_{and left for 7 days. Adjusting pH, obtaining}

dry biomass, taking samples and analysing samples were done according to same procedures mentioned in chapter 3.2.2. All experiments and analysis of samples were carried out in duplicates. To differentiate the flasks with the same media, the flasks were numbered.

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Distilled water 100 g/l

Yeast extract (OXOID, England) 1 g/l

Acetic acid (Merck, Germany) 0.2 g/l Yeast extract (OXOID, England) 1 g/l

Acetic acid (Merck, Germany) 0.2 g/l MgSO4 * 7H2O (BDH Laboratory supplies,

England)

0.75 g/l

3.2.4 Concentration of acetic acid in liquid media

Media described in Table 6 and 7 were prepared with the same procedures mentioned in chapter 3.2.2. Cultivations were carried out in 250 ml Erlenmeyer flasks with 100 ml working volume. The Erlenmeyer flasks were put in a linear shaking water bath at 30 °C with 118 oscillations min1_{and left for 7 days.}

Samples from each Erlenmeyer flasks were taken during the experiment to control pH and the concentration of acetic acid. A pH-meter was used to control pH and adjustments were conducted as described in chapter 3.2.2. All experiments were carried out in duplicates. To differentiate the flasks with the same media, the flasks were numbered.

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England)

0.75 g/l

England)

0.75 g/l

CaCl2 * 2H2O (Merck, Germany) 1.0 g/l

3.2.5 Analytical methods

Throughout the experiments, the concentration of acetic acid was determined by using gas chromatography. Displayed below in Figure 13, a standard curve used for calculating the concentration of acetic acid in the liquid media samples as mentioned in chapter 3.2.2 and 3.2.3.

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25 Figure 13. Standard curve of acetic acid from gas chromatography

The standard curve displayed in Figure 14 was used for calculating the concentration of acetic acid in liquid medium samples as mentioned in chapter 3.2.4.

Figure 14. Standard curve for liquid cultivation with different acetic acid concentrations from gas chromatography y = 180097x R² = 0,9902 0 10000 20000 30000 40000 50000 60000 70000 80000 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 Ar ea

Acetic acid standard concentration [g/L]

Standard curve of acetic acid from gas chromatography

y = 739265x R² = 0,9803 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 Ar ea

Acetic acid standard concentration [g/l]

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4 RESULTS AND DISCUSSION

During the preliminary study visual observations were conducted daily to compare the growth of R. oligosporus and M. indicus inoculated on PDA and MEA medium. Visual comparison showed that both culture media supported the growth of cultivated fungi. however, PDA performed better, as it is shown in Figures 15 and 16 below.

Figure 15. Comparison of growth of M. indicus on PDA and MEA, day 4

Figure 16. Comparison of growth of R. oligosporus on PDA and MEA, day 5

Comparing PDA and MEA as cultivation media for the fungi mentioned above, it was discovered that PDA as cultivation medium generally had less contamination as well as a better growth. The reason for that the fungi had better growth on PDA compared to that on MEA could be that the fungus had easier access to carbon from potato dextrose than from malt extract.

In addition, overheating MEA during sterilization might reduce the efficiency of the medium due to damaged properties caused by Maillard reaction. This might be one of the reasons why the fungi grew poorly in MEA. It is advised to sterilize MEA medium carefully and correctly to avoid overheating to prevent Maillard reaction (Crueger et al., 1990). However, further

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research needs to be performed to understand why PDA was preferable as growth medium. For further cultivations on agar plates, PDA was used as growth medium in this study.

The high rate of contaminated plates was mostly because of airborne spores of Aspergillus due to other students who worked with the fungi. Other contributing factors to contamination could be, the human factor as well as high humidity, high temperatures combined with the ease for insects to wander in the laboratory. As unsatisfactory growth of M. indicus was noticed, M. indicus was incubated at 28 °C instead of 37 °C in all further incubations.

Results of obtained standard curves (Figure 17 and 18), where the density for each spore solution was obtained by using haemocytometer. The density of the spore solution was then calculated according to the formula from Mikrobiologi Umum written by Pengajar (2013).

𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 = 𝑁𝑁𝑁𝑁. 𝑁𝑁𝑜𝑜 𝐷𝐷𝑠𝑠𝑁𝑁𝑠𝑠𝐷𝐷𝐷𝐷

𝑁𝑁𝑁𝑁. 𝑁𝑁𝑜𝑜 𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝐷𝐷𝐷𝐷 × 𝑉𝑉𝑁𝑁𝑉𝑉. 𝑁𝑁𝑜𝑜 𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝐷𝐷𝐷𝐷 × 𝐷𝐷𝐷𝐷𝑉𝑉𝑠𝑠𝐷𝐷𝐷𝐷𝑁𝑁𝐷𝐷 𝑜𝑜𝑠𝑠𝑓𝑓𝐷𝐷𝑁𝑁𝑠𝑠

Figure 17. Standard curve for R. oligosporus. Absorbance plotted against density of spore solution y = 6,18E-06x - 8,39E-03 R² = 9,99E-01 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14

0,00E+00 5,00E+03 1,00E+04 1,50E+04 2,00E+04 2,50E+04

Abs or ba nc e [ A]

Density of spore solution [spores/ml]

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Figure 18. Standard curve for M. indicus. Absorbance plotted against density of spore solution

R. oligosporus required shorter time for growth compared to that for M. indicus, this might be because R. oligosporus has a shorter generation time compared to other edible fungi as it requires a simple ecosystem to survive. This fungus can grow vigorously between 25 and 45 °C (Cantabrana et al., 2015). According to Babu et al. (2009), R. oligosporus produces natural, heat-stable antibiotic agents that could fight against bacterial diseases. This might also yield a faster growth of 4 days compared to 7 days for M. indicus.

However, when comparing standard curves of the fungi, it was noted that the density obtained at absorbance of 0.1 was 8.88 × 104_{spores per ml for M. indicus, whereas that for R.} oligosporus was 1.75 × 104_{spores per ml. Spore suspension of M. indicus contained more}

fungal spores than the spore suspension of R. oligosporus this is possibly because of the spore size differences. According to (Jennessen et al., 2008a, Chen et al., 2007), R. oligosporus has larger spore size compared to that of M. indicus. However, in this experimental study, M. indicus was recultivated three times on agar plates due to slow sporulation and contamination, whereas R. oligosporus was cultivated just once. Figure 18 shows the result from the successful sporulation of M. indicus. Overall, R. oligosporus grew better, faster and sporulated satisfactory on PDA growth medium.

y = 1,25E-06x - 1,10E-02 R² = 9,99E-01 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4

0,00E+00 5,00E+04 1,00E+05 1,50E+05 2,00E+05 2,50E+05 3,00E+05 3,50E+05

Abs or ba nc e [ A]

Density of spore solution [spores/ml]

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During this work, V. volvacea was also cultivated, but as a mushroom, it requires fruiting bodies in order to produce fungal spores (Bao et al., 2013). Due to this, spore suspension was not executed; hence a standard curve could not be created.

Figure 19 shows the results from cultivating R. oligosporus and V. volvacea in different liquid media. A comparison of growth, expressed as final dry weight, in a media containing acetic acid and a media containing minerals, yeast extract and acetic acid were conducted.

Figure 19. Comparison of final dry weight obtained with fungi cultivation in different media

According to the results shown on Figure 19, there was a minimal growth of R. oligosporus obtained even in the media containing only acetic acid and water. Biomass growth would require even other nutrients than only a carbon source, as acetic acid here, so the obtained small amount of biomass could be the consequence of possible contaminations presented providing traces of nutrients necessary for the growth. 0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04

Acetic acid, yeast extract and

minerals Acetic acid, yeast extract andminerals Acetic acid

V. volvacea R. oligosporus R. oligosporus

Dr y w ei gh t o f b io m as s [ g]

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The following images were taken from the last day of the cultivations (Figure 20).

a) b)

c)

Figure 20. Results in duplicate of liquid cultivation day 7. a) V. volvacea cultivated in acetic acid, yeast extract and minerals. b) R. oligosporus cultivated in acetic acid, yeast extract and minerals. c) R. oligosporus cultivated in acetic acid

Figure 21. Comparison of acetic acid concentration over time

Figure 21 shows the concentration of acetic acid obtained in the cultivation media over time. At day 3 V. volvacea cultivated in acetic acid, yeast extract and minerals have still levels of

0 0,05 0,1 0,15 0,2 0,25 0 2 4 6 8 Co nce nt ra tio n o f a ce tic aci d Day

Concentration of acetic acid over time

V. volvacea 1.Acetic acid, yeast extract and minerals

R. oligosporus Acetic acid, yeast extract and minerals R. oligosporus 1.Acetic acid

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acetic acid left in the cultivation media, why R. oligosporus has already consumed all acetic acid on day 3. This might indicate that R. oligosporus grows slower compared to V. volvacea. Moreover, considering that all acetic acid was already consumed within the first three days in some of the cultivations, samples should be taken more often during the first days of the cultivations to get accurate results and a complete picture on the consumption of acetic acid. Regarding the cultivation of R. oligosporus in only acetic acid (green line on Figure 13) there was a promising consumption initially and up to day 3. However, from day 3 to day 7 the concentration of acetic acid rather stabilized in the cultivation, with a slight increase observed. This might be because of selecting a wrong sample vial to analyse or an error in the gas chromatography measurements. To correct possible errors, duplicate analyses should have been made. Regardless of this, as it was discussed earlier, there should not be any

consumption of acetic acid at all in this cultivation, as fungi cannot grow solely on carbon, the presence of other nutrients are also necessary for the growth. These results might indicate that there was a kind of contamination present in the sample providing necessary nutrients for some growth during the first days. Although samples were taken daily, only day 1, 3 and 7 was analysed due to expensive costs for using gas chromatography.

Figure 22. Comparison of calculated biomass yield

Biomass yield was calculated by dividing dry weight of biomass produced with the amount of consumed acetic acid obtained during the cultivation, according to the formula written below:

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

1.Acetic acid, yeast extract and

minerals Acetic acid, yeast extract andminerals 1.Acetic acid

V. volvacea R. oligosporus R. oligosporus

Yi

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𝑠𝑠 =

𝑥𝑥 (𝑆𝑆0−𝑆𝑆)∗𝑉𝑉

there Y is yield (g/g), x is produced biomass (g dry weight), S0 and S are initial and final

substrate concentrations (g/l), respectively, and V is the working volume (L) of the cultivation.

Calculated biomass yield in Figure 22 showed that cultivation of V. volvacea gave the highest yield, and the next highest yield was obtained for R. oligosporus. In both cases the fungi were cultivated in a medium containing acetic acid, yeast extract and minerals. Seemingly, V. volvacea yields more biomass than R. oligosporus. However, when inoculating, V. volvacea, mycelia together with a piece of agar was used, while in case of R. oligosporus a spore solution was used. These different methods applied could cause the differences determined for the final dry biomass, thus giving a higher yield. To minimize the differences, inoculation should be practiced with the same method, when inoculating with R. oligosporus, cutting mycelia together with a piece of agar might have been a better method instead of using 1 ml of spore suspension as inoculum, to be able to control the amount of biomass used for the inoculation better.

The low yield from cultivation in only acetic acid is because of lack of nutrients, as there was no added minerals or yeast extract in the medium. Furthermore, using only acetic acid would cause low pH, which needs to be adjusted. During experiment only pH indicator strips were used as control when adjusting pH, hence keeping a constant pH was difficult. The small amount of biomass obtained in this cultivation might have occurred due to a possibility that the cells “eat each other” to gain nutrients or contamination has occurred.

In summary, it is evident from this study, that fungi can use acetic acid as a carbon source. Although using acetic acid solely in liquid cultivation led to a production of a small amount of biomass, other nutrients should also be provided for proper growth.

For optimization of fungal growth, different compositions of the liquid media as well as linear and orbital shaking baths were compared. As seen in Figure 23, cultivation in a medium containing acetic acid and minerals in linear shaking bath produced the highest amount of dry weight biomass among all the liquid media tested, while the medium containing only yeast extract produced the smallest amount of dry weight biomass. Additionally, it was also found that linear shaking bath performed better than orbital shaking bath.

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Figure 23. Comparison of dry weight obtained during different cultivation conditions

Figure 24. Comparison of acetic acid consumption over time

Figure 24 shows data on acetic acid consumption when comparing cultivation of R. oligosporus in different liquid media. The results show that all acetic acid was consumed in all cultivations at day 7. The fastest consumption rate was obtained when R. oligosporus was grown in acetic acid and minerals in an orbital water shaking bath (green line) there as soon as at day 1 was all acetic acid consumed. In the medium containing acetic acid and yeast extract (blue line) and in the medium containing acetic acid and minerals (purple line), both in linear

0 0,005 0,01 0,015 0,02 0,025

Linear Orbital Linear Orbital Orbital

Acetic acid and

yeast extract Acetic acid andyeast extract Acetic acid andminerals Acetic acid andminerals Yeast extract

Dr y wei gh t o f b io ma ss [g ]

Dry weight of R. oligosporus

0 0,05 0,1 0,15 0,2 0,25 0 2 4 6 8 Co nce nt ra tio n o f a ce tic aci d Day

Concentration of acetic acid over time

Acetic acid and yeast extract. Linear

2. Acetic acid and yeast extract. Orbital

2. Acetic acid and minerals. Orbital

3. Acetic acid and minerals. Linear

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water shaking bath, all acetic acid was consumed at day 3. However, at day 1 R. oligosporus in a medium containing acetic acid and yeast extract had consumed more acetic acid compared with that in a medium containing acetic acid and minerals. This might indicate an initially better growth in a medium with acetic acid and yeast extract due to more favourable conditions. Lastly, R. oligosporus cultivated in acetic acid and yeast extract in an orbital water shaking bath (red line), grew seemingly slowest as consumption of all acetic acid was finally made only at day 7.

As there are different outcomings regarding media composition as well as type of water shaking bath, it is hard to determine which of the media composition and type of water shaking bath would be preferable. Our results, show, that a medium of acetic acid and minerals in an orbital water shaking bath generated an environment where R. oligosporus could easily consume acetic acid (Figure 24). On the other hand, when using a linear water shaking bath, a medium of acetic acid and yeast extract seems to be preferable resulting in a faster growth. For a better comparison duplicate analyses as well as everyday analyses would be preferable in future experiments.

Figure 25. Comparison of biomass yield of R. oligosporus during different conditions

A relatively high value, among obtained yields, was gained during cultivation of R. oligosporus in acetic acid and minerals using a linear water shaking bath (Figure 25). As growth of the fungi is cotton like, it was hard to determine why the different results from

0 0,2 0,4 0,6 0,8 1 1,2 1,4

Linear Orbital Linear Orbital

Acetic acid and yeast

extract 2.Acetic acid and yeastextract 3.Acetic acid andminerals 2.Acetic acid andminerals

Yi

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cultivating in linear or orbital water shaking bath had occurred (Figure 26). One possible reason might be the influence of aeration on biomass production. Aeration is necessary aiming to produce higher biomass yield (Sablayrolles and Goma, 1984) therefore, the high biomass yield obtained when using linear water shaking bath indicates that the aeration in linear water shaking bath might be better than that in orbital water shaking bath. However, as there are different outcomings when comparing linear and orbital water shaking baths (Figure 25), further investigations are needed to conclude how linear and orbital shaking affects fungal growth. In all, according the results obtained here linear water shaking bath was used during the following cultivations conducted in this study.

a) b)

c) d)

e)

Figure 26. Results of duplicate liquid cultivations of R. oligosporus, at day 7. a) Acetic acid and yeast extract, linear shaker. b) Acetic acid and yeast extract, orbital shaker. c) Acetic acid and minerals, linear shaker. d) Acetic acid and minerals, orbital shaker. e) Yeast extract, orbital shaker

Prior to the next experimental setups, the results of obtained dry weight biomass of R. oligosporus produced at different cultivation conditions, shown on Figure 23, were evaluated.

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Although a medium consisting of only yeast extract produced biomass, this resulted in the lowest amount produced. This might be because of the lack of minerals, or easy utilization of carbon, as a medium consisting of both acetic acid and yeast extract as well as a medium consisting of acetic acid and minerals produced higher values of dry weight (Figure 23). Furthermore, as it is shown on Figure 22, R. oligosporus grow with higher yield on medium containing acetic acid, minerals and yeast extract. Thus, it was decided that further cultivations will be conducted with a medium consisting of acetic acid, minerals and yeast extract, since it could not be proved, from the results gained that a medium without yeast extract would be necessarily better than a medium with yeast extract.

During the last part of this study this medium composition was therefore used, however with different concentrations, i.e. 0.2 and 2.0 g/L, of acetic acid. As expected, in medium with higher acetic acid concentration higher amount of biomass could be produced (Figure 27).

Figure 24. Comparison of dry weight of R. oligosporus obtained during fungal cultivations at different acetic acid concentrations

Out of the two Erlenmeyer-flasks containing 2.0 g/l acetic acid, one of them produced a dry weight of 0.1518 g, the other Erlenmeyer-flask containing 2.0 g/l acetic acid produced a dry weight of 0.8615 g making a huge difference between the results obtained in parallel duplicate setups, causing a high measurement error as it is shown on Figure 27. However, observing the images, that were taken from the last day of cultivation (Figure 28), it was concluded that the

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1

2,0 g/l acetic acid, yeast extract and minerals 0,2g/l acetic acid, yeast extract and minerals

Dr y wei gh t o f b io ma ss [g ]

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obtained 0.8615 g dry weight was probably measured incorrectly. After reweighing the dry biomass, a second obtained weight of approximately 0.10 g could be considered as more correct.

a) b)

Figure 25. Results of duplicate liquid cultivation of R. oligosporus with different acetic acid concentration, day 7 a) Concentration of acetic acid: 0.2 g/l

b) Concentration of acetic acid: 2.0 g/l

Figure 26. Comparison of acetic acid consumption over time

Figure 29 shows how acetic acid was consumed over time, as seen, all acetic acid of the medium containing 2.0 g/l acetic acid (red line) was consumed within 5 days, the steep decrease of acetic acid from day 0 to day 2 indicates a rapid growth due to favourable

conditions. Further measurements would be of interest to gain more data between day 0 and 2 as well as day 2 and 5 where the consumption of acetic acid seemingly decreases.

From Figure 29 it was also notable that the medium containing 0.2 g/l acetic acid (blue line) had virtually consumed all acetic acid as soon as at day 2. This might indicate that an initial

0 0,5 1 1,5 2 2,5 0 2 4 6 8 Co nce nt ra tio n o f a ce tic aci d Day

Acetic acid concentration over time

Acetic acid, yeast extract and minerals. 0.2 g/l initial concentartion of acetic acid Acetic acid, yeast extract and minerals. 2.0 g/l initial concentartion of acetic acid

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concentration of 0.2 g/l acetic acid was not enough as carbon and energy source for a cultivation during 7 days. Further measurements as well as additional cultivations with concentrations of acetic acid amidst 0.2 g/l and 2.0 g/l as well as concentrations above 2.0 g/l acetic acid might be of interest in order to find an optimal concentration of acetic acid for fungi cultivation.

Figure 27. Comparison of calculated biomass yield from different acetic acid concentrations

Results of yield in Figure 30 clearly shows that a medium containing 2.0 g/l acetic acid gives a higher yield. However, as discussed above, the error in obtained weight of dry biomass would affect the value of the presented yield of the cultivation in 2.0 g/l acetic acid.

If the faulty weight is discarded, the new calculated yield from cultivating in 2.0 g/l acetic acid would be lower than the obtained yield from the cultivation in 0.2 g/l acetic acid.

This could mean that acetic acid might act as an inhibitor for fungal growth, 0.2 g/l in a fed-batch system might be a better alternative as higher concentrations of acetic acid seems to act inhibitory on fungal growth. However, further investigations are needed to confirm this assumption.

5 CONCLUSIONS

PDA medium is an excellent growth medium to be utilized for the maintenance of fungal strains. All cultivations carried out in liquid media produced more or less biomass regardless of the composition of the medium. It was proved that acetic acid in general seems to be a

0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 0.2 g/l 2.0 g/l

Acetic acid, yeast extract and minerals Acetic acid, yeast extract and minerals

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suitable carbon and energy source for fungi to utilize. However, acetic acid alone is not enough, a combination of acetic acid, yeast extract and/or minerals is a preferable medium composition, since it supplies sufficient nutrients for fungal growth. Determining preferred type of water shaking bath was difficult, as both linear and orbital water shaking baths had mixed results, further research regarding this area might be of interest. Nevertheless, further research for optimum concentration of acetic acid as well as cultivation mode in reactors could be the focus in future studies when investigating acetic acid as carbon and energy source.

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Edible Fungal Production using Acetic Acid as Carbon and Energy Source

E

DIBLE

F

UNGAL

P

RODUCTION USING

A

CETIC

A

CID AS

C

ARBON AND

E

NERGY

S

OURCE

Abstract

Sammanfattning

TABLE OF CONTENTS

1. INTRODUCTION

1.1 Background

1.2 Aim and objective

1.3 Research questions

2. LITTERATURE STUDY

2.1 Edible fungi

2.2 Volatile fatty acids

2.3 Factors influencing aerobic submerged cultivation

3 MATERIAL AND METHODS

3.1 Material

3.2 Method

Standard curve of acetic acid from gas chromatography

4 RESULTS AND DISCUSSION

Concentration of acetic acid over time

Dry weight of R. oligosporus

Concentration of acetic acid over time

Acetic acid concentration over time

5 CONCLUSIONS

REFERENCES