Bio hydrogen bio butanol generation by bacteria isolated from spent mushroom substrate

BIO-HYDROGEN AND BIO-BUTANOL GENERATION BY BACTERIA ISOLATED FROM SPENT MUSHROOM SUBSTRATE MARC-ANTOINE METAIS (GRADUATE STUDENT OF LYCEE SAINTE GENEVIEVE, PARIS DAUPHINE UNIVERSITY AND ECOLE CENTRALE PARIS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgment I would like to thank my supervisor, Professor He, who has helped me in this research. I appreciate her time and willingness to share her ideas and opinions with me. I would also like to express my deep appreciation for the time, effort and energy that my mentor, Xin Fengxue, invested in my research. He was always insightful, supportive and understanding. His expertise and ability to guide, listen and provide feedback contributed enormously to the quality of my research. Fengxue provided invaluable advices and was always willing to provide me with needed assistance. I feel extremely fortunate for having Xin Fengxue as my mentor. I am also thankful for the support that my family, friends and labmates provided me during my study: Yu Ran, Becky, Xiaomei, Constance, Chenxi, Shen Yujia, Yan Yu, Si Yan, Cheng Dan, Dr Chua, Dr Ray, Dr Li, Qi Chao, Lam Yuen Sean, Ding Chang, Wang Shan Quan, Lim Kah Bin, Thierry Desmarest, Kukik, Nur, Alexandre and Victor. Unquestionably, without their support I would not have been able to complete this study. Because of this, I would like to dedicate this dissertation to all of you who made it possible. i TABLE OF CONTENTS Ackowledgment........................................................................................................i Abstact......................................................................................................................v List of figures.........................................................................................................vii List of tables………….............................................................................................x List of symbols………………………………………………….………………...xi CHAPTER 1. Introduction and Literature Review.............................................1 1.1 Spent Mushroom Substrate............................................................................1 1.2 Value-added products.....................................................................................5 1.2.1 The potential of hydrogen....................................................................8 1.2.2 Butanol and its use.............................................................................11 CHAPTER 2. Methodology..................................................................................15 2.1 Medium Composition...................................................................................15 2.2 Medium Making Protocol............................................................................16 2.3 Inoculation....................................................................................................17 2.4 Biological Safety Cabinet………………………………………………….19 2.5 Analysis........................................................................................................20 2.5.1 Gas analysis…………………………………………………….…...20 2.5.2 Gas Chromatography – Thermal Conductivity Detector………..….21 2.5.3 Gas Chromatography – Flame Ionization Detector……………...….22 2.6 DNA Analysis – Strain Identification……………………………………..24 ii CHAPTER 3. Isolation of bacteria from Spent Mushroom Substrate............27 3.1 Growth of mixed culture on Spent Mushroom Substrate.............................27 3.2 Growth of mixed culture on glucose............................................................31 3.3 Growth of mixed culture on different substrates..........................................36 3.4 Isolation of pure cultures using glucose as a carbon source.........................39 3.5 Identification of strains S4 and S11………………………….……………41 CHAPTER 4. Growth of bacteria using different kinds of carbon sources....................................................................................................................47 4.1 Growth of pure cultures using glucose as a carbon source..........................47 4.2 Growth of pure cultures using xylose as carbon source……………….…..51 4.3 Growth of pure cultures using Spent Mushroom Substrate as carbon source………………………………………………………………………...…...55 4.4 Pre-treatment of Spent Mushroom Substrate...............................................62 4.4.1 Enzymes production...........................................................................62 4.4.2 Enzymes purification..........................................................................63 4.4.3 Hydrolysis....................................... ..................................................70 4.4.4 Fermentation.......................................................................................71 4.5 Glucose concentration optimization.............................................................72 4.6 Upscaling of the laboratory-scale experiments............................................74 4.6.1 Protocol…………………………………………..…………………74 4.6.2 Results................................................................................................78 iii CHAPTER 6. Conclusion.....................................................................................83 6.1 Major findings……………………………………………...……………...83 6.2 Recommendations and future studies…………………................………...84 References..............................................................................................................86 iv Abstract The world is now being overwhelmed by many kinds of waste. Agricultural waste is a major issue, especially in emerging countries, where there is usually no recycling facilities. This study focuses on Spent Mushroom Substrate, which is the compost material remaining after a few cropping cycles of the mushroom industry. The main objective of our study is to isolate and cultivate bacteria from this Spent Mushroom Substrate. An anaerobic fermentation process producing bio-hydrogen and bio-butanol is studied, using classic carbohydrates such as glucose, xylose, cellulose and xylan, and then Spent Mushroom Substrate as carbon sources for the micro-organisms. Few previous studies were done on this topic. In this study, three strains of bacteria were isolated from Spent Mushroom Substrate, able to generate value-added products from simple carbohydrates (predominantly glucose). A full DNA analysis was conducted for the two best strains. We further performed a thorough study of these bacteria growing on several kinds of Spent Mushroom Substrate, in order to assess which kind of Spent Mushroom Substrate could be used efficiently. Then, an optimization process of the experiment conditions was conducted in order to improve the output of the experiments. The substrate concentration was optimized, and a pre-treatment of the Spent Mushroom Substrate, before the fermentation, was also performed. The last part of the study is a pre-industrial simulation. We upscaled the laboratory experiment in order to test whether the process could be done on-site, directly in a mushroom farm. v One strain was particularly good in generating bio-hydrogen and bio-butanol, and was named S11. However, most of Spent Mushroom Substrates were not easily degraded by S11. This is due to the molecular structure of Spent Mushroom Substrate we tested: it can be made of very different kinds of materials (horse manure, different kinds of wood sawdust…), come from very different places (China, Malaysia…), and be stored in different conditions (temperature, humidity, contamination…). After optimization of the fermentation process of S11 on glucose, the molar percentage of hydrogen was about 30%, and the butanol concentration was about 6.0 g/L, which are high concentrations. We noted that the pre-treatment process of Spent Mushroom Substrate was not efficient enough, and therefore not costeffective for a mushroom farm. Finally, the pre-industrial simulation was successful. We proved that upscaling the experiment did not cause any major disturbance on the fermentation process: the results are very similar to those obtained when using laboratory bottles. However, the process is slower than when using laboratory bottles: this would be an issue on a practical viewpoint. As a conclusion, we managed to isolate a good hydrogen–producing and butanolproducing strain, S11, from Spent Mushroom Substrate. The output of the anaerobic fermentation is high concentrations of hydrogen and butanol, which are highly-regarded value-added products nowadays. A pre-industrial simulation was conducted and showed that the process we designed has potential to be implemented on-site. vi List of figures Figure 1 Top production of Mushroom and Truffles – Source: [1] ..........................1 Figure 2 Energy content of different chemicals. Source: [19] ..................................9 Figure 3 Biochemical Pathways in Clostridium acetobutylicum during acidogenic phase - Source [47] .................................................................................................14 Figure 4 Orbital Shaker Incubator ..........................................................................18 Figure 5 Gas Chromatocgraphy Analyzer, Agilent Technologies ..........................22 Figure 6 Microscope view of Oyster Mushroom and Lentinula Edodes endogenic mixed culture ..........................................................................................................28 Figure 7 Gas production of OM mixed culture growing on OM ............................29 Figure 8 Gas production of LE mixed culture growing on LE ..............................30 Figure 9 Total gas production of mixed cultures growing on glucose 30g/L .........32 Figure 10 Gas composition of OM mixed culture growing on glucose 30g/L .......33 Figure 11 Gas composition of LE mixed culture growing on glucose 30g/L .........33 Figure 12 Biosolvents production of OM mixed culture growing on glucose 30g/L (mM) .......................................................................................................................34 Figure 13 Biosolvents production of LE mixed culture growing on glucose 30g/L (mM) .......................................................................................................................35 Figure 14 Total gas production of mixed cultures growing on different carbon sources.....................................................................................................................37 Figure 15 Biosolvents production of mixed cultures growing on different carbon sources.....................................................................................................................38 Figure 16 Anaerobic Chamber ................................................................................40 Figure 17 Colonies on agar plate ............................................................................41 vii Figure 18 Picture after DGGE (from left to right: S4, ladder, S11) .......................42 Figure 19 Extract of DNA Sequence for S4 Strain .................................................43 Figure 20 Extract of DNA Sequence for S11 Strain ...............................................43 Figure 21 Phylogenetic tree for S4 Strain ...............................................................44 Figure 22 Phylogenetic tree for S11 Strain .............................................................45 Figure 23 Cumulative gas production of pure cultures growing on glucose 30g/L 48 Figure 24 Biosolvents production of S4 pure culture growing on glucose 30g/L ..49 Figure 25 Biosolvents production of S11 pure culture growing on glucose 30g/L 50 Figure 26 Biosolvents production of ESE1 pure culture growing on glucose 30g/L .................................................................................................................................50 Figure 27 Cumulative gas production of pure cultures growing on xylose 30g/L .52 Figure 28 Biosolvents production of S4 pure culture growing on xylose 30g/L ....53 Figure 29 Biosolvents production of S11 pure culture growing on xylose 30g/L ..53 Figure 30 Biosolvents production of ESE1 pure culture growing on xylose 30g/L .................................................................................................................................54 Figure 31 Spent Mushroom Substrate : from the farm to dried powder .................56 Figure 32 Total gas production of S4 growing on SMS 30g/L...............................57 Figure 33 Total gas production of S11 growing on SMS 30g/L.............................58 Figure 34 Fermentation bottles ...............................................................................59 Figure 35 Biosolvents production of S4 growing on A 30g/L................................59 Figure 36 Biosolvents production of S4 growing on OMN 30g/L .........................60 Figure 37 Biosolvents production of S11 growing on A 30g/L..............................60 Figure 38 Biosolvents production of S11 growing on OMN 30g/L .......................61 Figure 40 Beaker with xylanase solution ................................................................64 Figure 41 Dialysis process ......................................................................................65 viii Figure 42 Freeze-drying of the enzymes ................................................................66 Figure 43 Test tubes for optical density measurement ...........................................68 Figure 44 Spectrophotometer samples ....................................................................69 Figure 45 Standard curve for optical density of enzymes .......................................70 Figure 39 Gas composition of S11 pure culture growing on glucose at different concentrations at pH=5.5 ........................................................................................73 Figure 46 Bio-fermenter and gas tank at the initial step .........................................75 Figure 47 Bio-fermenter and gas tank after 12 hours .............................................76 Figure 48 Total gas production of S11 pure culture growing on glucose 30 g/L on pre-industrial scale ..................................................................................................78 Figure 49 Gas composition of S11 pure culture in Fermenter ................................79 Figure 50 Biosolvents production (mM) of S11 pure culture in Fermenter ...........80 ix List of tables Table 1 Components of Spent Mushroom Substrate. Source: [12]..……………….3 Table 2 PCR conditions …………………………………………………………..25 Table 3 Hydrogen yield of S4, S11 and ESE1 on Glucose 30 g/L ……………….49 Table 4 Standards composition …………………………………………………...67 x List of symbols SMS – Spent Mushroom Substrate OM – Oyster Mushroom LE – Lentinula Edodes A – Abalone Mushroom GC – Gas Chromatography FID – Flame Ionization Detector TCD – Thermal Conductivity Detector PCR – Polymerase Chain Reaction DGGE – Denaturing Gradient Gel Electrophoresis xi CHAPTER 1. Introduction and Literature review 1.1 Spent Mushroom Substrate According to the Food and Agriculture Organization of the United Nations [1], the global production of cultivated edible mushrooms has increased from 0.79 tons in 1970, to 2.07 million tons in 1990 and 6.53 million tons in 2009. It is expected that this amount will increase in the future due to market demand. In 2009, Singapore mushroom production was 18 tons. About 53% of cultivated edible mushrooms are produced in Asian countries (China being the largest producer), followed by European countries (32%) and the Americas (13%). In Asia, mushrooms are eaten and appreciated for their flavor, and used medicinally for their healing properties. Figure 1 Top production of Mushroom and Truffles – Source: [1] 1 After a few cropping cycles (usually 2 to 4), the mushroom productivity diminishes, and the compost material is called “spent”, and it is then replaced by fresh compost. Despite the evident benefits of mushrooms, the exponential increase in their consumption worldwide is also generating a high volume of spent mushroom substrate (SMS). Spent Mushroom Substrate is discarded and treated as waste. It has been reported that about 5 kg of substrate are needed to produce 1 kg of mushroom [2], and about 30 million tons of SMS are produced each year. SMS has a storage problem because it is wet and putrefies quickly. Consequently, one of the main problems faced by mushroom production companies is finding a way to properly dispose of the SMS without contaminating the water and soil. In fact, the lack of a sustainable waste management solution for SMS is the most significant barrier to the future development of the mushroom industry [3]. Several studies have been carried out to demonstrate the benefits of SMS application in mushroom re-cultivation, enrichment of soils, restoring areas that have been destroyed through environmental contamination [4], cultivation of vegetables, fruits and flowers in greenhouses and fields [5], use as animal feed [6] and soil amendment and degradation of organopollutants [7]. The SMS can also be used as a potential energy feedstock [8], and ethanol production [9]. Thus, environmental concerns have been escalating recently concerning its effective disposal and recycling: in China, wastes are largely burnt by the farmers, which causes air pollution issues. Mushroom producers use specially formulated compost as growth medium to cultivate their mushrooms: the major ingredients used to make the substrate are straw, hay, corncobs, horse manure, poultry litter, 2 gypsum, lime. Among them, one of the most common sources is wood sawdust, which is routinely used for the cultivation of king Oyster Mushroom (Pleurotus eryngii) and Winter Mushroom (Flammulina velutipes). Spent mushroom substrate is a nutrient-rich organic product. The chemical composition of SMS determines its potential for reuse and environmental effects. On average, fresh SMS is about 60% water and 40% dry material by mass [10], whereas approximately 65% of the dry matter is organic and 35% is inorganic salts [11]. Generally speaking, SMS contains high levels of organic, C, N, and inorganic Ca+, Mg2+, Na+, K+, Cl-, SO42- . Components Content (g/kg) Organic C 290 to 340 Organic N 17 to 26 P 5 to 7 S 50 to 60 K 21 to 26 Ca 83 to 97 Na 2 to 4 Mg 5 to 8 Cl 6 to 8 Table 1 Components of Spent Mushroom Substrate. Source: [12] 3 In this study, we use the Spent Mushroom Substrate from an organic mushroom farm located in Johor, Malaysia. The SMS was used to harvest several species of mushroom, mainly Pleurotus Ostreatus (OM, Oyster Mushroom), Lentinula Edodes (LE, Shiitake), and Pleurotus cystidiosus (A, Abalone Mushroom). Oyster mushroom is a mushroom species commonly found in Asia, is edible and is believed to have medicinal virtues: it contains statins which work to reduce cholesterol. Lentinula Edodes (Shiitake, LE) is also an edible mushroom native to East Asia, and is used as a delicacy as well as a medicinal mushroom, in this region. Abalone Mushroom is native to and still found growing wild in China. The Abalone mushroom is named for the aquatic shellfish, abalone, whose shape the mushroom resembles. It can be used for its bioremediation properties of organopollutants. The SMS from this mushroom farm was stored in anaerobic condition in a sealed plastic bag. The time-duration of one mushroom culture cycle is about 4 months. Then the anaerobic plastic bags were brought to our laboratory one month after the harvest of the mushroom. Spent Mushroom Substrate was made of different materials, depending on the mushroom to be grown on it. For OM, SMS was made of oak tree sawdust, from China. For A, SMS was made of rubber tree sawdust, from Malaysia. This mushroom farm is an organic mushroom farm, so the only component of the SMS is wood sawdust: no chemical was added to the SMS. Moreover, we can assume that the SMS is very clean, since, in order to grow the mushroom mycelium, there has to be no contamination. 4 After harvest, mushroom have degraded part of the sawdust, producing carbohydrates, constituting the major carbon source for the bacteria. Therefore, there are two contradictory phenomena occurring at the same time. The more we wait, the more wild bacteria will grow without any control and consume the carbohydrates in the SMS. On the other hand, the more we wait, and the more the mycelium will produce carbohydrates by degrading SMS. After a given period of time, the former phenomenon will be predominant, because there is a fixed amount of biomass that can be degraded by the mycelium. A typical mushroom farm, such as the one we worked with, generates about 15 tons of SMS per month, which is 180 tons per year. This SMS has to be disposed of, and most companies cannot do it by themselves, spending money on waste management. The mushroom farm we worked with being an organic mushroom farm, they convert most of their SMS into organic compost, to grow other on-site agricultural products. Our aim is to provide a process which could essentially help mushroom farms not to pay for SMS disposal, and turn this waste into value-added products. 1.2 Value-added products According to the second assessment report of the Intergovernmental Panel on Climate Change (IPCC) [13], the Earth’s surface temperature has increased by about 0.2ºC per decade since 1975. Furthermore, recognizing a number of uncertainties, “the balance of evidence suggests that there is a discernible human 5 influence on global climate” as the result of activities that contribute to the production of greenhouse gases. By preventing heat radiated from the sun-warmed earth from escaping into space, the increased concentration of greenhouse gases in the atmosphere contributes to climate change. The gases that produce the greenhouse effect are carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and a host of engineered chemicals such as hydrofluorocarbons (HFCs) and perflorocarbons (PFCs). About 90% of U.S. greenhouse gas emissions from anthropogenic sources come from energy production and use [14], and most (82%) of these emissions are a byproduct of the combustion of fossil fuels. CO2 accounts for a majority of recent increases in the heat-trapping capacity of the atmosphere, with worldwide atmospheric concentrations of CO2 increasing at about 0.5% annually. Anthropogenic CO2 has resulted in atmospheric CO2 concentrations that exceed preindustrial levels by 30%. Energy-efficient, renewable-energy, and other low-carbon technologies reduce CO2 emissions by reducing the need for fossil fuel combustion. Significant reductions in greenhouse gas emissions can be accomplished only through an assemblage of actions ranging from more effective production, distribution, and use of energy to a reliance on lower-carbon fuels. Given the magnitude of carbon emission reductions needed to stabilize atmospheric CO2 concentrations, multiple approaches to carbon management will be needed. Such changes have the potential to transform the world’s buildings, industries, vehicles, and electricity production. Each of the three energy end-use sectors (buildings, industry, and transportation) account for approximately one-third of CO2 6 emissions. This diversity of sources and uses of fossil energy means that no single technological “fix” exists for reducing carbon dioxide emissions. Using the framework of the 11-Lab study [15], there are three options for reducing atmospheric carbon. First, energy efficiency can decrease the energy intensity of the world economy, thereby reducing carbon emissions. Energy-efficient technologies and products such as more efficient cars, trucks, and household appliances provide the same energy services using less fuel or electrical power and thereby emitting less carbon. Similarly, energy requirements can be reduced through efficient system designs, such as co-locating facilities that produce both electrical power and heat with facilities that need them. A broad array of energy-efficiency options exists. Second, the use of low-carbon technologies can decrease the carbon intensity of the world’s energy economy, thereby reducing carbon emissions. These technologies either increase the efficiency of energy production or use fuels that emit less carbon such as renewable energy resources and nuclear power. Electricity generation from natural gas is also a low-carbon technology when compared to current coal-fired power plants; natural gas emits 13 MtC per quad of energy used compared with 25 MtC per quad for coal [16]. Biomass feedstocks offer an array of low-carbon options, including ethanol fuels, chemicals, materials, and electricity. The carbon emissions from biomass combustion are largely offset by CO2 absorption during plant growth. 7 Third, carbon sequestration technologies offer another suite of approaches to reducing atmospheric concentrations of CO2. Carbon sequestration can include various ways of removing CO2 from the atmosphere and storing it, or keeping anthropogenic carbon emissions from reaching the atmosphere by capturing and diverting them to secure storage [17]. Most approaches to carbon sequestration will require considerable additional research to ensure their successful development and acceptance. However, in the long-term, they could play significant roles. This thesis focuses on the second point: the use of low-carbon technologies. We emphasize the need to generate value-added by-products from clean sources, such as bacterial fermentation. We will highlight the importance of hydrogen and butanol, both produced by either a mixed culture of bacteria or single-strain colony, isolated from Spent Mushroom Substrate. 1.2.1 The potential of hydrogen Hydrogen is considered to be one of the most promising fuels of the future [18] due to its high energy content, 130 kJ/g, as compared to hydrocarbon fuels. 8 Figure 2 Energy content of different chemicals. Source: [19] Hydrogen gas is also a clean fuel with no COx, SOx and NOx emissions from its combustion. Besides, hydrogen is an important energy carrier and can be used in fuel cells for generation of electricity [20]. However, hydrogen gas is not readily available in nature like fossil fuels and natural gas, but can be produced from renewable materials such as biomass and water [21]. Hydrogen gas production technologies have gained special attention during the last fifty years due to the increasing energy demand, rapid consumption of non-renewable fossil fuel reserves and hydrocarbon fuel based atmospheric emissions. Users regularly purchasing pressurized hydrogen gas do it in heavy steel cylinders containing about 0.6 kg of H2 per cylinder. The price of this hydrogen has been reasonably stable at about $100/kg, plus cylinder rental [22]. 9 Steam reforming of natural gas and water electrolysis are the most commonly used processes for H2 gas production. Due to the energy intensive nature of those processes, more energy efficient H2 production methods are searched for. Hydrogen gas production from renewable resources such as biomass, and carbohydrate rich waste materials by bioprocesses, offers distinct advantages over energy intensive methods used. Major drawbacks in biohydrogen production are low yields and productivities requiring large reactor volumes and long residence times [23]. As mentioned beforehand, nowadays, hydrogen is usually produced by thermochemical processes, electro-chemical processes and bio-conversion processes. Among them, bioconversion process is a promising method for two reasons: utilization of renewable resources, and usually operated at ambient temperature and atmospheric pressure. Main bio-hydrogen production processes are direct or indirect bio-photolysis, dark and photo-fermentations. Bio-photolysis of water under sunlight is considered as the cleanest approach for bio-hydrogen production. However, low H2 gas productivity, strict light requirement and oxygen inhibition are the main problems in bio-photolysis of water [24]. Fermentative hydrogen gas production from carbohydrates is a much faster process than bio-photolysis, with volatile fatty acids (VFAs) and H2 gas formation [25]. However VFAs need to be fermented for further H2 gas production [26]. Major mechanisms for bio-hydrogen production by fermentation have been 10 elucidated. However, development of an effective bio-hydrogen production process at industrial scale is still a challenge. Up to now, the reported rates and yields of fermentative hydrogen gas production were not high enough to make the process economically viable. The most suitable raw materials, pre-treatment methods, bacterial cultures, operating conditions, cultivation types, operating modes and processing schemes are yet to be determined for an effective and economically viable fermentative hydrogen production process. 1.2.2 Butanol and its use The second byproduct we want to highlight is butanol C4H9OH. Though ethanol C2H5OH and biodiesel are driving the world’s progress on the road of renewable fuels, some attention is being generated by butanol, a fuel whose promoters believe can become a real player in the world of energy alternatives. Butanol is a 4-carbon alcohol: C4H9OH. Today, butanol is typically produced from petroleum sources, but that has not always been the case. During the first half of the 20th century, the production of butanol from biological sources was a commercial reality. According to the National Renewable Energy Laboratory (NREL), biobutanol had previously been produced through a fermentation process known as “ABE” named 11 such because it produced Acetone, Butanol, and Ethanol in roughly 6:3:1 ratios. Clostridium strains were the fermenting organisms to create the chemicals from molasses-type feedstocks [27]. This ABE process nearly disappeared in the 1960s because it could not compete on a cost basis with the economical creation of solvents from cheap, plentiful petroleum. The current market for butanol is largely industrial, for use as a plasticizer or solvent. The world market for butanol is estimated at 350 million gallons per year, with the U.S. accounting for about 220 million of that. The average cost for a gallon of butanol is between three and four U.S. dollars [28]. Butanol has been generating some attention as a potential alternative fuel, akin to ethanol but with added benefits. One of those qualities is butanol’s low Reid vapor pressure (RVP), a rating of 0.33 psi versus ethanol’s 2 psi and gasoline’s 4.5 psi, meaning butanol has lower evaporative emissions [29]. Another property is its energy content. Butanol has a higher energy density (29.2 MJ/L) than ethanol (19.6 MJ/L), but lower than gasoline (32 MJ/L) [30]. So, switching a gasoline engine over to butanol would in theory result in a fuel consumption penalty of about 10%. However, using butanol instead of ethanol as an alternative fuel would result in a consumption benefit of 48%. This figure shows how efficient would butanol be compared to ethanol, if we were to switch from gasoline engines to biofuel engines. Butanol can be blended at higher concentrations in gasoline than existing biofuels without modifications to automotive engines. Butanol is considered substantially 12 similar to gasoline for blending purposes and is certified by the U.S. Environmental Protection Agency as a blending agent up to 11 percent [31]. Currently, no production vehicle is known to be approved by the manufacturer for use with 100% butanol. As of 2009, only few vehicles are approved for even using E85 fuel (i.e. 85% ethanol + 15% gasoline) in the USA. However, in Brazil all vehicle manufacturers (Fiat, Ford, VW, GM, Toyota, Honda, Peugeot, Citroen) produce flex fuel vehicles that can run on 100% ethanol or any mix of ethanol and gasoline. These flex fuel cars represent 90% of the sales of personal vehicles in Brazil, in 2009. BP and Dupont, engaged in a joint venture to produce and promote butanol fuel, claim that "biobutanol can be blended up to 10%v/v in European gasoline and 11.5%v/v in US gasoline" [31]. According to analysis done by the National Renewable Energy Lab [32], most of the advantages of butanol come from its properties as a fuel, not from current production technology. Traditionally, low yields - in the 15 to 25 percent range have plagued butanol production from bacterial reactions. Toxicity is also a problem, with butanol generally being toxic to microbes at the rate of 20 g/L. 13 Figure 3 Biochemical Pathways in Clostridium acetobutylicum during acidogenic phase - Source [47] 14 CHAPTER 2. Methodology All experiments were conducted under the same experimental conditions, unless otherwise stated. All experiments were duplicated in order to have a higher accuracy for the results. 2.1. Medium composition The anaerobic bacteria used in all the following experiments were grown in a liquid medium. This liquid medium is named modified DCB1, unless otherwise stated. It provides pH-buffers and elements which are necessary to the growth of our cultures [48], [49], [50]. In every cases, the medium used to grow the cultures of bacteria is the same. The modifications are the following: 1- TES (pKa=7.55) is not used, but instead we use MES (pKa=6.15): 2-(Nmorpholino)ethanesulfonic acid: 10 mM. MES is more suitable because its pH buffer range is relevant to the pH of the reactions required (around 6.0). 2- We add KH2PO4: 0.5 g/L 3- We add K2HPO4: 0.5 g/L 4- We add Yeast extract: 5 g/L 2.2 Medium Making Protocol 15 The protocol for making modified DCB1 medium is very strict, because the anaerobic property must be strictly observed all along the making of the medium. Following is the protocol used for the experiments in this paper. The volume of medium prepared is 1800 mL, so all the amounts are given for 1800 mL of modified DCB1 solution. Rinse rubber stoppers with deionized-H2O and dry them with scientific cleaning wipes. Then, place them in clean beaker, cover with aluminum foil and autoclave. This step is meant to prevent the rubber stoppers from contamination. The following step is to prepare defined mineral salt medium. Using a 2-L flask, measure 1800 mL of deionized-H2O with 10 mL salt solution, 1 mL trace element solution, 1 mL Se/W solution, and 0.25 mL resazurin. Put the flask onto an electrothermal heating mantle and set up refrigerated bath circulator and condenser. Bring to boil for 20 minutes under N2-purging at 1 psi. After 20 minutes boiling, cool solution to room temperature under N2-purging at 2 psi in ice-water bath. Introduce new N2-source and switch off refrigerated bath circulator and condenser. Measure buffering agents and reductants while solution cools: MES 10 mM, 0.0242 g L-Cysteine, 0.048 g Na2S.9H2O, 0.0771 g DTT, K2HPO4 0.5 g/L, KH2PO4 0.5 g/L, Yeast extract 5 g/L. Add the buffering agents and reductants while stirring with a magnetic stir bar. Insert pH-probe into solution and adjust pH to 7.0 by flushing with N2 and careful addition of 10% H2SO4 or 20% NaOH. Flush 60-mL syringe with the headspace 16 gases 2 to 3 times and aliquot 35 mL of solution into the bottles, under N2-purging for 1 to 2 minutes, stopper and seal bottle with crimper after 1 to 2 minutes. Ensure all bottles are properly labeled, then autoclave for 20 minutes at 121°C. After autoclave, all the bottles were stored at air temperature, and kept ready for addition of carbon source and inoculation. 2.3 Inoculation Inoculation was done inside of the anaerobic chamber. Anaerobic chamber provides a strict anaerobic atmosphere of 0-5 parts per million using a palladium catalyst and hydrogen gas mix of 5%. In order to use the chamber, one has to bring in a baked catalyst, an anaerobic indicator (to test O2 level before starting the experiment) and an empty beaker for condensed water every time entering the chamber. The condition to use the anaerobic chamber is to minimize the O2 input. If items with sealed bags or caps (e.g., syringes larger than 3 mL, vials/tubes larger than 2 mL) are to be placed into the chamber, one has to slightly tear the bag open or loosen a bit the caps of vials/tubes, in order to let O2 out during the autocycle. Autocycle consists of a 3 times successive vacuum and N2 injections, inside of the airlock, in order to make sure that the airlock is made anaerobic. After that, the chamber itself, when bringing the items from the airlock to the chamber, can be considered anaerobic. 17 The first step of inoculation is to remove the cap of the bottle, and scoop the defined amount of carbon source (glucose or SMS most of the time, in this study) into the 1st-generation autoclaved bottles. The second step is to inject the bacteria into the bottle. Use sterile syringe and needle to inject the colony into the bottle. Usually, in this study, and unless otherwise stated, the inoculation will be done at 5% (v/v). The last step is to stopper and seal the bottle with a crimper. Finally, bring bottles out of the anaerobic chamber, use a syringe to release the gas (N2 flushed during medium preparation) contained in the headspace of the bottles, and place in orbital shaker incubator at 35°C and 150 rpm. Figure 5 Orbital Shaker Incubator All the following experiments in this study were conducted with an additional control bottle. This control bottle is not mentioned in the results to make this paper 18 more clear for the reader. However, for every experiment, a control bottle was prepared with the same substrate as in the experiment, but without any inoculation of bacteria. This control bottle was prepared in order to detect a possible contamination. When gas or biosolvents were detected in the control bottle, the experiment was re-conducted. This step is of paramount importance in order to ensure that our results are as accurate as possible. 2.4 Biological Safety Cabinet A Biological Safety Cabinet is an enclosed, ventilated laboratory workspace for safely working with materials potentially contaminated with pathogens requiring a defined biosafety level. The Biological Safety Cabinet used in our laboratory is a ThermoScientific Herasafe KS, with a safety level of Class II. The US Centers for Disease Control and Prevention defines Class II Biological Safety Cabinets as hoods used to protect personnel, product and environment from bio-aerosols and other particulates. These hoods offer personnel protection through engineered airflow into the cabinet. To protect the product, the work area in the cabinet is continuously bathed with ultra-clean air provided through the supply HEPA filter. Approximately 70% of the air from each cycle is recirculated through this supply HEPA filter [46]. Before and after using a Biosafety Cabinet, the working surface was disinfected with a 70% alcohol solution. Materials for the experiment were placed inside the cabinet prior to the start of the experiment. Overloading of material in the cabinet 19 should was avoided because materials may obstruct optimal airflow. We allowed two minutes of purge time after placing materials in the cabinet. When performing an experiment, materials were kept away from the front air intake and rear exhaust. Transfer of viable materials were performed as deeply into the cabinet as possible. The cabinet was never turned off while a flame is being used. Following completion of the experiment, the cabinet was allowed to purge itself for about two minutes. 2.5 Analysis All samples were analysed on an everyday basis (unless otherwise stated). Three different kinds of measures will be presented in this part. Unless otherwise stated, all the graphs in this study are done until day 13. The reason is that 13 days after inoculation, all the experiments had no gas production anymore, and there was no more change in the biosolvents concentrations (including butanol). 2.5.1 Gas analysis The first analysis that was automatically conducted is the gas release from the bottles. The protocol for this analysis is very simple: apply ethanol on the stopper, gently shake the bottle, insert needle and syringe, and allow the gas to fill the syringe. Then, remove the syringe when the piston reaches equilibrium level, dispense gas in the fume-hood. Reinsert the syringe into the bottle in case the 20 volume of gas produced is more than the syringe volume. Finally, the amount of gas produced is noted down every day. 2.5.2 Gas Chromatography - Thermal Conductivity Detector The second analysis is to measure the gas composition. We used a GC-TCD (Gas Chromatography - Thermal Conductivity Detector), which is a non-specific and non-destructive detector. The GC-TCD is based on the principle of thermal conductivity which depends upon the composition of the gas. The sample components in the carrier gas pass into the measuring channel. A second channel serves as a reference channel where only pure carrier gas flows. Electrically heated resistance wires are located in both channels. The difference in thermal conductivity between the column effluent flow (sample components in carrier gas) and the reference flow of carrier gas alone, produces a voltage signal proportional to this difference. The signal is proportional to the concentration of the sample components. Like all chromatographic analytical processes, gas chromatography is a relative method: calibration with a standard mixture is required, both to check linearity and as calibration for the sample. 21 Figure 6 Gas Chromatocgraphy Analyzer, Agilent Technologies The GC-TCD used was manufactured by Agilent Technologies. The protocol for this analysis is to apply ethanol on the stopper, gently shake the bottle, insert needle and small syringe, and extract a volume of gas of 100 L from the fermentation bottle. Then, inject the gas onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column was already calibrated to measure concentrations of H2, N2 and CO2. CH4, which is also a good source of biofuel, was also produced by the fermentation process, but this study focuses on H2 only. 2.5.3 Gas Chromatography - Flame Ionization Detector The third analysis is to measure the organic compounds concentrations, using GCFID (Gas Chromatography – Flame Ionization Detector). In GC-FID, the FID 22 detects analytes by measuring an electrical current generated by electrons from burning carbon particles in the sample. The FID is a non-selective detector, so there is a potential for many non-target compounds present in samples to interfere with this analysis and for poor resolution especially in complex samples. The FID works by directing the gas phase output from the column into a hydrogen flame. A voltage of 100-200V is applied between the flame and an electrode located away from the flame. The increased current due to electrons emitted by burning carbon particles is then measured. Although the signal current is very small (the ionization efficiency is only 0.0015%) the noise level is also very small. Except for a very few organic compounds (e.g. carbon monoxide) the FID detects all carbon containing compounds. In our experiments, we focus on butanol, acetic acid, and butyric acid. Most of the time, ethanol and acetone will also be generated, and sometimes, propionic acid is produced in reasonable amount. However, we do not mention it in this study since the main focus is butanol. All the graphs shown below emphasize butanol, acetic acid and butyric acid only, I order to make the graphs clearer for the reader. The GC-FID used was manufactured by Agilent Technologies. To prepare the samples, one need to use the Biological Safety Cabinet. The first step is to dispense 25 L of 1 mol/L Sulfuric Acid H2SO4 in a 2 mL microfuge tube, and then to add 0.475 mL of the sample to be tested. Then, vortex the sample and centrifuge it at 10°C, 14,000 rpm for 20 minutes. Finally, aspirate 420 L of the supernatant, dispense into a 2 mL screw cap vial, and tighten screw cap. Place the tubes for reading by the GC-FID. 23 2.6 DNA Analysis – Strain Identification The process for strain identification is in several steps. Cells (1 mL) used for DNA extraction were withdrawn from cultures and then centrifuged immediately at 14,000 rpm for 15 minutes at 4°C. After removing the supernatant, the cell pellets were stored at -20 ºC until further processing. The extraction of the genomic DNA of the strains was done using DNeasy Blood and Tissue Kit (250), by Qiagen GmbH, Germany, according to the manufacturer’s instructions. Polymerase chain reactions (PCR) were carried out in an Eppendorf Master Cycler ep gradient S thermocycler (Eppendorf AG, Hamburg, Germany). The extracted DNA was used as a template for Polymerase Chain Reaction (PCR) amplification of the 16S rRNA gene, which were amplified with a pair of universal bacterial primers 8F (Forward: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1392R (Reverse: 5'-ACGGGCGGTGTGT-3'). The reaction mixture (50 µl) contained 5×PCR buffer, 2.5 mmol/L MgCl2, 0.13 mmol/L BSA, 0.25 mmol/L dNTP, 1.25 U of Taq DNA polymerase, 0.05 µmol/L forward primer and 0.05 µmol/L reverse primer, and 20 ng template DNA. 24 Process Temperature °C Duration Initial Denaturation 95 2'10'' Denaturation 95 30'' 55 45'' Extension 72 2'10'' Final Extension 72 6' Annealing 30 cycles Table 2 PCR conditions The PCR-amplified 16S rRNA was purified and its size was verified by low melting point agarose electrophoresis. TAE buffer was prepared by mixing 40 mL of TAE solution in 1960 mL of Milli-Q ultra-pure water. 1 gram of Agarose powder (SeaKem LE Agarose, BioWhittaker Molecular Applications, USA) was mixed with 100 mL of 1xTAE mixture in a conical flask. The Agarose powder was thoroughly dissolved by heating up in microwave for about 90 seconds. Dissolved Agarose was poured into a casting tray to allow solidification. The gel casting tray was first leveled using a bubble level and its well comb placed securely before the gel was poured in and allowed to harden. Trapped air bubbles in the hardening gel were removed using a pipette tip so that they would not affect DNA migration during electrophoresis. Comb was removed once gel is hardened. Gel was then transferred, together with the tray, into the electrophoresis unit. 1xTAE buffer was poured into the tray to cover the gel completely. Thawed extracted DNA samples were vortexed and centrifuge in micro-centrifuge tubes. 5 μL of 100 bp or 1-kb DNA ladder (Promega, Madison, US) was then loaded into 25 the first well. 1 μL of 6xblue/green loading dye (Promega, USA) was mixed with 5 μL of a DNA sample and then loaded into a well. Electrophoresis (Bio-Rad, U.S) was done at 90V for 90 minutes when all DNA samples had been loaded. Gels were removed from tray The bands in gel were visualised by UV excitation and pictures were taken with a digital camera (Gel Doc, Bio-Rad, USA). 26 CHAPTER 3. Isolation of bacteria from Spent Mushroom Substrate The purpose of this study is to cultivate bacteria generating value-added products for on-site production. So the emphasis is now put on finding, isolating and using a particular strain, or consortium of strains, isolated from SMS and generating valueadded products (here, we focus on hydrogen and butanol). Several steps were undertaken. The first one was to grow the mixed culture directly from SMS on different carbon sources. The second one was to grow isolated pure cultures from SMS on different carbon sources. The carbon sources used in this paper are glucose, xylose, cellulose, xylan, and SMS itself (2 different kinds). SMS should be particularly hard to degrade, according to its complex composition. 3.1. Growth of mixed culture on Spent Mushroom Substrate This first experiment was run in order to find out whether the bacteria (mixed culture) growing on SMS can grow by themselves, using SMS as a carbon source, to generate value-added products. No other carbon source was added in this experiment, in order to be as accurate as possible, regarding the overall amount of SMS consumed. The SMS was added fresh into the DCB1 medium, so the carbon 27 source is similar to the one found in the mushroom farm, after the mushroom are grown and harvested. The SMS used is the one to grow the two mushroom strains OM and LE, as previously stated. A preliminary microscope screening shows the presence of a lot of different bacterial strains in both bacterial sources (OM and LE). Several bacterial morphologies can be observed, such as spheres (coccus), or round-ended cylinders (bacillus). We can also notice different aggregation properties (bacteria density) among these two samples. This diversity is a good indicator of the growth of bacteria in the SMS after the harvest of mushroom. We will try to isolate interesting strains from these samples, in order to generate value-added products. Figure 7 Microscope view of Oyster Mushroom and Lentinula Edodes endogenic mixed culture For this first experiment, the pH was controlled on a daily basis in order to replicate the natural living conditions of the bacteria. The initial pH after inoculation was 5.5; hence, everyday, the pH was adjusted to 5.5, using either 28 H2SO4 or NaOH solutions. Several SMS concentrations in the medium were used: 2.5 g/L, 5 g/L, 10 g/L, and 15 g/L, for both OM and LE substrates. The results of the gas production for both OM and LE are presented here. 30 25 V o 20 l u m 15 e Day 13 Day 5 Day 4 Day 3 ( m 10 L Day 1 ) 5 0 OM-2.5g/L OM-5g/L OM-10g/L OM-15g/L Figure 8 Gas production of OM mixed culture growing on OM 29 30 25 Day 13 V o 20 l u m 15 e Day 5 Day 4 Day 3 ( m 10 L ) Day 1 5 0 LE-2.5g/L LE-5g/L LE-10g/L LE-15g/L Figure 9 Gas production of LE mixed culture growing on LE As can be seen on these graphs, the gas production is low (less than 30 mL after 13 days of fermentation). The hydrogen concentration was also low: about 0.5 mM for all the samples. The conclusion is that mixed culture in the SMS cannot grow very well by itself on the SMS in modified DCB1 medium. Additional carbon source needs to be given, and pure cultures need to be isolated, in order to improve the fermentation process. 30 3.2 Growth of mixed culture on Glucose This second experiment was run in order to find out whether the bacteria (mixed culture) growing on SMS can grow using glucose as a carbon source, to generate value-added products. The pH was controlled on a daily basis in order to replicate the natural living conditions of the bacteria. The initial pH was 5.5 after inoculation; hence, everyday, the pH was set to 5.5, using either H2SO4 or NaOH solutions. The first step for this experiment was to prepare and isolate the mixed culture. A 5% (w/v) inoculation of fresh OM and LE SMS was done in a 30 mL bottle of modified DCB1 medium with 30 g/L glucose. The incubation was done in a 35°C shaker, 150 rpm. The gas produced by the bacteria was released every day, for 13 consecutive days. After 13 days, there was no more gas production, and the concentration of glucose in the solution was close to 1.2 g/L, meaning almost all the glucose was used by the bacteria. The colonies in fresh SMS can therefore degrade glucose very well. At the end of the previous experiment, 5% (v/v) of the previous bottle was inoculated in another 30 mL bottle of DCB1 medium, with 30 g/L of glucose, for both OM and LE mixed culture. This step is meant to cultivate the mixed culture, and to constitute a stock-bottle of this mixed culture for future experiments. The culture was incubated in the 35°C shaker, 150 rpm, for 13 days. At this stage, the only carbon source was glucose 30 g/L: we can assume there was no leftover 31 from the previous culture because the glucose measured after 9 days was close to 0 g/L, and the new inoculation wass done at 5% (v/v). The daily gas production was recorded for 13 days. The results are shown below: “LE” and “OM” being the names given to the mixed cultures isolated from OM and LE SMS. LE OM 0 20 40 60 80 100 120 Volume (mL) Figure 10 Total gas production of mixed cultures growing on glucose 30g/L As can be seen on this graph, the results are very similar for the two different kinds of SMS. The total gas production after 13 days is close to 120 mL. These results are much more convincing than the previous study, where the only carbon source was SMS itself. This track should be further studied. 32 25 ( C o 20 n c e 15 n m t M r 10 a t 5 i o n 0 H2 ) N2 CO2 1 5 9 13 Time (day) Figure 11 Gas composition of OM mixed culture growing on glucose 30g/L 20 18 ( C o n c e n m t M r a t i o n 16 14 12 10 H2 8 N2 ) CO2 6 4 2 0 1 5 9 13 Time (day) Figure 12 Gas composition of LE mixed culture growing on glucose 30g/L 33 As can be seen on these diagrams, the molar percentage of hydrogen increases over time, and goes from 29% one day 1 to 36% on day 13 for OM; and from 12.5% on day 1 to 38% on day 13 for LE. So, the highest molar percentage of hydrogen is found after 13 days of inoculation. The amount of glucose remaining after 13 days was measured for OM and was 12.4 g/L. We can therefore calculate the yield of hydrogen production for this reaction, which is: H2 yield(mol hydrogen produced / mol glucose consumed) = 0.651 This yield, compared to existing papers, is relatively high [38]. Regarding the biosolvent production, the concentrations were measured 13 days after inoculation. Acetic acid, butyric acid and butanol were detected for both OM and LE mixed cultures. 23.6 42 Acetic Acid Butyric Acid Butanol 62 Figure 13 Biosolvents production of OM mixed culture growing on glucose 30g/L (mM) 34 40 52 Acetic Acid Butyric Acid Butanol 24.8 Figure 14 Biosolvents production of LE mixed culture growing on glucose 30g/L (mM) As can be seen on the graphs above, butyric acid and butanol constitute the major part of the biosolvents production. Butanol concentration is 3.11 g/L for OM mixed culture and 3.85 g/L for LE mixed culture. The previous results imply that some of the bacterial strains in OM and LE mixed cultures are potentially good candidates to produce high amounts of hydrogen and butanol. Up to now, we know that OM an LE mixed culture can grow well on glucose. In order to use SMS as a carbon source, further studies should be done on whether this mixed culture can degrade more complex carbohydrates, such as xylose, cellulose and xylan. 35 3.3 Growth of mixed culture on different substrates This third experiment was run in order to find out whether the mixed cultures growing on spent OM substrate can grow using not only glucose as a carbon source, but also xylose, cellulose and xylan, to generate value-added products. If so, then we can be confident that these mixed cultures will be able to degrade SMS, whose structure and composition are much more complex. The OM mixed culture used in this experiment is the same as in the previous experiment. A first inoculation was done at 5% (v/v) from the mixed culture stockbottle to a modified DCB1 bottle, in order to make sure that there was no glucose leftover from the stock-bottle. Then, another 5% (v/v) inoculation was done to a modified DCB1 bottle with only one carbon source, stated later for each particular bottle. Glucose and xylose were added at a concentration of 30 g/L (liquid phase) directly into the modified DBB1 bottle. Cellulose and xylan from birchwood had to be prepared in two steps, since the commercial products are in solid phase. The final concentration in the modified DCB1 medium was 30 g/L. The cultures were incubated in the 35°C shaker, 150 rpm, for 13 days. At this stage, the only carbon source is the one stated for each bottle: glucose, xylose, cellulose or xylan; there is no leftover from the stock bottle because two 5% (v/v) inoculations were done successively in order to dilute the potential carried-over glucose from the seed culture bottle. The pH was controlled on a daily basis in 36 order to replicate the natural living conditions of the bacteria. The initial pH after inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H2SO4 or NaOH solutions. 140 120 V 100 o l u 80 m e 60 Day 13 ( Day 1 m L Day 9 Day 5 40 ) 20 0 xylose glucose cellulose xylan Figure 15 Total gas production of mixed cultures growing on different carbon sources This graph shows that the mixed cultures from OM SMS can grow well on different substrates. The best results are for glucose 30 g/L, and cellulose is the substrate which is the hardest to be degraded. The total gas production for all the carbon sources is non negligeable: we thus can be confident that this mixed culture can grow reasonably well on SMS as a carbon source. Regarding the gas composition, hydrogen had a molar percentage from 10% to 30% for all carbon 37 sources, which is consistent with the previous experiments. Following are the results for the biosolvents production after 13 days. 140 ( 120 C o n 100 c e 80 n m t M r 60 a t 40 i o n 20 Butanol Butyric Acid ) Acetic Acid 0 xylose glucose cellulose xylan Figure 16 Biosolvents production of mixed cultures growing on different carbon sources Again, glucose is the best carbon source for the mixed culture from OM SMS. Xylose is also well degraded by the mixed culture, but in much lower quantities. However, cellulose used as a carbon source doesn’t give any detectable amount of butanol. Therefore, it is predictable that SMS could be degraded quite well by OM mixed culture: the four carbon sources used here are already degraded by OM mixed culture. 38 We now know that some of the strains both in OM and LE can produce hydrogen and butanol in non-negligeable amounts. In order to get better results, we need to isolate the pure cultures and to further experiment on these strains. 3.4 Isolation of pure cultures using glucose as a carbon source The focus of this step is to isolate pure cultures from SMS. We need to isolate the hydrogen and butanol producing strains, and then to improve their fermentative efficiency, by optimizing the experiment conditions. All the following manipulations were done in an anaerobic chamber. The previous bottles were kept in order to store the mixed cultures from OM and LE SMS. Then, agar plates were prepared, using glucose 30 g/L as carbon source. Several dilution factors of the inoculate were tested in order to get the broadest possible range: 1/10; 1/100; 1/1,000; 1/10,000; 1/100,000 and 1/1,000,000. 0.2 mL to 0.5 mL liquid solution from OM and LE mixed cultures stock-bottles were spread to different agar plates, with the stated dilution factors. Agar plates were left in the anaerobic chamber for incubation. Single colonies started to appear about 3 days later. 39 Figure 17 Anaerobic Chamber The best dilution factor for our samples, both for OM and LE SMS was 1/100,000. Then, 20 positive colonies were picked up for each of the two bacterial sources (OM and LE), and transferred to modified DCB1 medium bottles, with glucose 30 g/L. These 40 new bottles were put in the shaker 35°C, 150 rpm, for 13 days. Gas was released and tested every day; and biosolvent analysis was run at the end of the 13 days, in order to discriminate the strains. Most of the strains couldn’t produce any butanol, and their hydrogen concentration was not significant. The full list of graphs for this experiment is not given here, since most of the strains were not of any interest for our study. 40 Figure 18 Colonies on agar plate Three strains were eventually selected among the 40, regarding their good potential to produce high volume of gas, high hydrogen concentration, and butanol. These three potentially good strains were named ESE1, S4 and S11. The next step is to use these pure cultures on 30g/L glucose in order to test their capability to degrade glucose and produce value-added products. 3.5 Identification of strains S4 and S11 As seen in the previous experiments, S4 and S11 are the two strains that seem to have the best potential for our study. The following experiments were conducted using strains S4 and S11: ESE1 was not used anymore. We therefore conducted 41 DNA analysis, according to the protocol detailed in 2.5, of these two strains in order to identify them. Figure 19 Picture after DGGE (from left to right: S4, ladder, S11) The first part of the protocol is to carefully follow the instructions of 2.5. The final step is to send our purified DNA samples (about 1,300 bp long) for sequencing to Applied Biosystems Company, together with our 8-F primer. Following are the results from this company. 42 Figure 20 Extract of DNA Sequence for S4 Strain Figure 21 Extract of DNA Sequence for S11 Strain 43 Figure 22 Phylogenetic tree for S4 Strain 44 Figure 23 Phylogenetic tree for S11 Strain 45 The National Center for Biotechnology Information website [45] suggests that Clostridium beijerinckii NCIMB 8052 is the closest strain to both S4 and S11. However, the similarity percentage is 98% for S11 strain, and 99% for S4 strain. We can therefore conclude that S4 and S11 are Clostridium sp. Further analysis should be conducted in order to get a 100% accurateness regarding the DNA sequence-matching with a known-microorganism. The objective of this part was to conduct a DNA analysis of the two best strains we have isolated. S4 and S11 are genetically similar. Although this is not the purpose of this study, a genetically engineered microbe (based on S11 genome, for instance), could be implemented in order to improve the performance of the fermentation process. 46 CHAPTER 4. Growth of bacteria using different kinds of carbon sources This part is the core part of our study. In the previous chapter, we isolated three strains (pure cultures) from Spent Mushroom Substrate in order to use them to produce value-added products: bio-hydrogen and bio-butanol. This chapter will focus on the degradation of several substrates by ESE1, S4 and S11. 4.1 Growth of pure cultures using glucose as a carbon source This experiment was conducted with the same experiment conditions as the previous ones. Every bottle was duplicated and the cultures put in the shaker 35°C, 150 rpm. Gas was released and tested every day; and a biosolvent analysis was run in order to discriminate the strains. The pH was controlled on a daily basis in order to replicate the natural living conditions of the bacteria. The initial pH after inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H 2SO4 or NaOH solutions. 47 400 350 V 300 o l 250 u m 200 e S4 S11 ( 150 ESE1 m L 100 ) 50 0 1 2 4 6 8 11 Time (day) Figure 24 Cumulative gas production of pure cultures growing on glucose 30g/L This graph clearly shows good results, considering the previous experiments done in this study. The total gas volume goes from 297 mL for ESE1 strain to 375 mL for S11 strain. Again, the hydrogen molar percentage was comprised between 10% (for ESE1) and 30% (for S11). The amount of glucose remaining after 11 days was measured for the three different strains. We can therefore calculate the yield of hydrogen production (mol hydrogen produced / mol glucose consumed) for this reaction. 48 H2 yield(mol hydrogen produced / mol glucose consumed) S4 0.56 S11 0.68 ESE1 0.51 Table 3 Hydrogen yield of S4, S11 and ESE1 on Glucose 30 g/L This yield, compared to existing papers, is relatively high [38]. In particular, S11 should be further studied. Then, the production of biosolvents was tested for every strain. This step is crucial to assess the potential of these strains to degrade Spent Mushroom Substrate. 120 ( C o 100 n c 80 e n m 60 t M r a 40 t i 20 o n 0 Butanol Acetic acid ) Butyric Acid 0 1 4 Time (day) 6 11 Figure 25 Biosolvents production of S4 pure culture growing on glucose 30g/L 49 140 ( C 120 o n 100 c e 80 n m t M 60 r a t 40 i o 20 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 26 Biosolvents production of S11 pure culture growing on glucose 30g/L 100 C o n c e n t r a t i o n 90 80 70 60 Butanol 50 Acetic acid 40 Butyric Acid 30 20 ( m M 10 ) 0 0 1 4 6 11 Time (day) Figure 27 Biosolvents production of ESE1 pure culture growing on glucose 30g/L 50 These three graphs demonstrate that the three strains generate butanol, which is very promising for our SMS substrate. For S11, butanol concentration after 11 days is 6.0 g/L. For S4, butanol concentration after 11 days is 5.48 g/L. For ESE1, butanol concentration after 11 days is 3.33 g/L These results are higher than the butanol concentration measured when using the mixed cultures from SMS on the same substrate (glucose 30 g/L). Nonetheless, further optimization will be done using this strain in order to test its ability to produce more butanol. Interestingly, butyric acid is generated in high quantity for S4, S11 and ESE1. Its concentration is 9.3 g/L when the strain is S11: this result is particularly interesting because the only carbon source here is xylose [44]. This is not the focus of our study, but S11 could be further studied to improve the butyric acid production, by optimizing the fermentation process. 4.2 Growth of pure cultures using xylose as a carbon source This experiment was conducted with the same experiment conditions as the previous one. Every bottle was duplicated and the cultures put in the shaker 35°C, 150 rpm. Gas was released and tested every day; and a biosolvent analysis was run in order to discriminate the strains. The pH was controlled on a daily basis in order to replicate the natural living conditions of the bacteria. The initial pH after inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H 2SO4 or NaOH solutions. 51 250 200 V o l 150 u m e 100 S11 S4 ESE1 ( m L ) 50 0 1 2 4 6 8 11 Time (day) Figure 28 Cumulative gas production of pure cultures growing on xylose 30g/L Then, the production of biosolvents was tested for every strain. This step is crucial to assess the potential of these strains to degrade Spent Mushroom Substrate. Following are the results for the 3 pure cultures growing on xylose 30 g/L. 52 70 ( C 60 o n 50 c e 40 n m t M r 30 a t 20 i o 10 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 29 Biosolvents production of S4 pure culture growing on xylose 30g/L 80 ( C 70 o 60 n c 50 e n m 40 t M r 30 a t 20 i o 10 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 30 Biosolvents production of S11 pure culture growing on xylose 30g/L 53 60 ( C 50 o n c 40 e n m 30 t M r a 20 t i o 10 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 31 Biosolvents production of ESE1 pure culture growing on xylose 30g/L These three graphs demonstrate that the three strains generate butanol, which is promising for our SMS substrate. For S4, butanol concentration after 11 days is 4.0 g/L. For S11, butanol concentration after 11 days is 4.8 g/L. For ESE1, butanol concentration after 11 days is 2.5 g/L. These concentrations are lower than when using glucose as a carbon source, which is consistent. It has to be noted that the concentration is still high, even if the molecular structure of xylose makes it harder to degrade than glucose. 54 4.3 Growth of pure cultures using Spent Mushroom Substrate as carbon source This experiment is to test the degradation of SMS 30g/L, and the following experiments are to change the parameters of the experiments in order to optimize the value-added products generation. The carbon sources from Spent Mushroom Substrates were used were named A (standing for Abalone Mushroom) and OMN (standing for Oyster Mushroom New). The initial preparation process is important, because, as seen in the previous chapter, fresh SMS is not a good substrate for bacteria. The first step of the initial preparation process is to dry the fresh SMS at 60°C in a oven. The fresh SMS was manually shredded, spread on a tray, and this tray was put in the 60°C oven for 3 days. After 3 days, the moisture of the SMS was evaporated, the SMS can be called “dried”. This dried SMS was then blended using a kitchen-mixer in order to make SMS powder, to get smaller dust particles, which are easier to be degraded by the bacteria. This powder was then autoclaved in the modified DCB1 medium bottles. Autoclave is of important since we want to avoid bacterial contamination: the product generation should come exclusively from our isolated strains, and not from any other unknown bacterial or fungal source growing in the fresh SMS. 55 Figure 32 Spent Mushroom Substrate : from the farm to dried powder This first experiment is meant to give a rough idea of what the potential of SMS as carbon source is. This experiment was conducted with the same experiment conditions as the previous ones (5% (v/v) inoculation). Every bottle was duplicated and the cultures put in the shaker 35°C, 150 rpm. Gas was released and tested every day; and biosolvent analysis was run in order to discriminate the strains and SMS. 56 The pH was controlled on a daily basis in order to replicate the natural living conditions of the bacteria. The initial pH after inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H2SO4 or NaOH solutions. The SMS concentration for this experiment was set to 30 g/L, to be consistent with the previous experiments, where glucose and xylose concentrations were 30 g/L. The main difference is that we do not know the composition of SMS. So it will not be possible to assess our results regarding the yield of the experiment. The only interpretable results of this experiment is its output: hydrogen production and butanol production. This is the main concern of the mushroom farm: they do not mind about the yield of the experiment, they want to generate value-added products from their waste. 40 35 V 30 o l 25 u m 20 e A OMN ( 15 m L 10 ) 5 0 1 2 4 6 8 11 Time (day) Figure 33 Total gas production of S4 growing on SMS 30g/L 57 35 30 V 25 o l u 20 m e 15 A OMN ( m 10 L ) 5 0 1 2 4 6 8 11 Time (day) Figure 34 Total gas production of S11 growing on SMS 30g/L As can be seen on these graphs, the total gas production is lower than when the carbon source is glucose or xylose 30 g/L. However, the total volume remains reasonably high: between 28 mL and 35 mL. The best strain is S11, followed by S4. This “ranking” of the strains is similar to when the carbon source was glucose or xylose 30 g/L. 58 Figure 35 Fermentation bottles 16 ( C 14 o n 12 c e 10 n m 8 t M r 6 a t 4 i o 2 n 0 Butanol ) Acetic acid Butyric Acid 0 1 4 6 11 Time (day) Figure 36 Biosolvents production of S4 growing on A 30g/L 59 ( 16 C 14 o n 12 c e 10 n m 8 t M r 6 a t 4 i o 2 n 0 Butanol ) Acetic acid Butyric Acid 0 1 4 6 11 Time (day) Figure 37 Biosolvents production of S4 growing on OMN 30g/L 16 ( 14 C o 12 n c 10 e n m 8 t M r 6 a t 4 i o 2 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 38 Biosolvents production of S11 growing on A 30g/L 60 16 ( C 14 o n 12 c 10 e n m 8 t M r 6 a t 4 i o 2 n Butanol ) Acetic acid Butyric Acid 0 0 1 4 6 11 Time (day) Figure 39 Biosolvents production of S11 growing on OMN 30g/L According to these results, A and OMN SMS do not seem to have a very high potential for butanol generation (the butanol concentration reached after 13 days is about 0.1 g/L for these SMS species). We decide to continue all the following experiments using S11 pure culture alone because S11 seems to be the most promising strain to generate value-added products. More results regarding biohydrogen and bio-butanol productions by S11 will be shown further on. These intermediate results are to be considered carefully. We need to take into account that further optimization will be done: the use of enzymes to pre-treat SMS (hydrolysis). 61 4.4 Pre-treatment of Spent Mushroom Substrate This step is meant to pre-treat Spent Mushroom Substrate in order to generate simple carbohydrates from SMS, which are easier to be degraded by strain S11. We use a xylanase-producing strain isolated from SMS. Xylanase is an enzyme able to degrade xylan, a complex sugar which is a major component of SMS. Four main steps were undertaken: enzymes production, enzymes purification, hydrolysis and fermentation. 4.4.1 Enzymes production The first step is xylanase generation, using microbial anaerobic fermentation. In order to produce enzymes able to degrade SMS, we use the same medium as usual, modified DCB1. The carbon source for the bacteria is xylan birchwood 10 g/L. The strain used to produce xylanase was isolated by my colleague Xin Fengxue, from OM Spent Mushroom Substrate. After further study by my colleague, genome identification and 16s rRNA analysis, this strain suitable for enzymes production, is Kluyvera sp. The fermentation process is the same as previously stated. Ten bottles with 50 mL medium and xylan as carbon source were prepared anaerobically, then a 5% (v/v) inoculation was done. Fermentation was done in the shaker 35°C, 150 rpm. Gas produced by the culture was released every day and noted down. No analysis of the gas composition or biosolvents concentrations were made, since this is not the 62 focus of this step. Nine days after inoculation, the fermentation process was stopped: xylanase was fully generated by the fermentation process. Then, the second step, enzymes purification could be undertaken. 4.4.2 Enzymes purification This second step doesn’t need anaerobic conditions, because xylanase doesn’t need anaerobic conditions. Thus, the ten 50 mL bottles containing the enzymes were open and the 500 mL liquid was aliquoted into a beaker. Electronic charges interactions are an issue for enzymes purification. The free charges in the medium are an obstacle for the enzymes to act as catalyzers: in order to solve this issue, ammonium sulfate H8N2O4S was added to the liquid, to annihilate the positive and negative charges in the medium. The beaker was installed in a 4°C cold room with a magnetic stirring bar. A 30% (w/v) amount of solid ammonium sulphate, 150 g in our experiment, was added to the 500 mL culture medium with constant stirring for 2 hours. The liquid was then centrifuged down at 9,000 rpm for 15 minutes and the precipitate was discarded. The supernatant was subsequently adjusted to 70% (w/v) saturation with addition of 200 g of ammonium sulphate, with constant stirring for 12 hours at 4°C in the cold room. 63 Figure 40 Beaker with xylanase solution After 12 hours, the liquid medium was centrifuged down again, the supernatant was discarded and the precipitate, which contains xylanase, was dissolved in a small volume of 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0. The xylanase solution was then subjected to dialysis for 24 hours at 4 °C against 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0. Dialysis is a diffusion process, which describes the property of substances in water which tend to move from an area of high concentration (our liquid medium containing enzymes) to an area of low concentration (the glycine buffer). Three intermittent changes of the buffer were done, after 2 hours, 6 hours and 13 hours, in order to improve the efficiency of the process, by lowering the concentration of dissolved 64 substances in the glycine buffer. After this step, the sample collected from the dialysis is made of pure enzymes. Figure 41 Dialysis process In order to further improve the purification process, we also carried out a freezedrying of the sample, because it still contains a relatively high amount of water, compared to the amount of (solid) enzymes isolated. Freeze-drying, or 65 lyophilization, is a dehydration process. It works by freezing the material, and then reducing the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase. In our case, the surrounding pressure was reduced to 0.06 millibar, for 31 hours. After this step, there should not remain any more water in the beaker, and the remaining solid phase is pure xylanase. Figure 42 Freeze-drying of the enzymes 66 Enzyme activity analysis was carried out from the dialyzed and freeze-dried sample. Enzyme activity was measured using the optical density method (with a spectrophotometer), and compared with the calibration curve obtained with standard test tubes. In order to achieve this, 6 standard test tubes were prepared, using the 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0. For every standard test tube, 1.0 mL of 1% (w/v) xylan solution (in the 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0) was added to the test tube. Then a volume of xylose stock solution 10 g/L and glycine buffer were added to the test tube in volumes stated in the following Table 2. The total volume of solution in a standard test tube was therefore 1.5 mL. Total xylose concentration is the standard which is going to be calibrated with the spectrophotometer, and compared with our enzymes sample. Xylose concentration (g/L) St0 0 St1 Xylose stock (mL) Glycine buffer (mL) 0 0.5 1.0 0.05 0.45 St2 2.0 0.10 0.4 St3 3.3 0.17 0.33 St4 5.0 0.25 0.25 St5 6.7 0.33 0.17 Table 4 Standards composition 67 The sample to be tested was prepared by mixing 1.0 mL of 1% (w/v) xylan solution, in the 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0, with 0.5 mL of sample (containing the purified enzymes) in a test tube. Two test tubes with our sample were prepared in order to duplicate the test, the total amount of solution in each test tube being 1.5 mL. The procedure for the test, similarly done for the standard test tubes and for our samples, is the following. The first step is to incubate the 1.5 mL mixture at 50°C for 10 minutes in a hot water-bath. Then the test tubes were placed on ice for 5 minutes. In every tubes, 3.0 mL of DiNitroSalicylic acid (DNS) solution was added. Then, the test tubes were incubated in a hot water-bath at 90°C for 10 minutes. When removed from the hot water bath, they were placed on ice again, for 20 minutes. Then, 20 mL of deionized water was poured in every test tube. Figure 43 Test tubes for optical density measurement 68 At this stage, we can visually compare the standard tubes with our sample, in order to have an idea of the concentration of reductive sugars in our sample. The darker the solution, the higher the concentration of reductive sugars: DNS reacts with reducing sugars to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. For more accurateness, and in order to get the exact enzyme activity, we use the spectrophotometer at 540 nm. Figure 44 Spectrophotometer samples The spectrophotometer gives the absorbance of every standard test tube at 540 nm. This absorbance was noted down to get a standard calibration curve. 69 8 7 6 5 ( 4 3 ) X y l o s e C o n c e n g t / r L a t i o n 2 1 0 0 0.172700003 0.241799995 0.309299991 0.362299994 0.476400003 Absorbance Figure 45 Standard curve for optical density of enzymes The mean absorbance for our sample is 0.25692. Reading on the standard curve for our sample gives us a xylose concentration of 3.612 g/L. One unit (U) of enzyme activity is defined as the amount of enzyme which liberate 1 μmol of product from their respective substrate per minute. This calculation is fully detailed in [42]. We can therefore calculate the U for our sample, which is 1.79/g substrate. 4.4.3 Hydrolysis The first two steps of the pre-treatment process, enzymes production and enzymes purification, are now completed. The third step is hydrolysis of Spent Mushroom Substrate. 70 Enzymatic hydrolysis was carried out at a substrate loading of 2.0% (w/v) with 10 IU xylanase/g of substrate in 20 mL of DCB1 medium adjusted to pH=8.0. The experiment was performed in duplicates at 55°C in a shaking hot-water bath for 72 hours. After hydrolysis, samples were centrifuged at 10,000 rpm for 10 minutes. The supernatant, containing simple carbohydrates was then collected. This liquid solution is to be the bacterial growth medium in the following fermentation process. 4.4.4 Fermentation The final step, after the pre-treatment process, is fermentation. The carbon source is made of simpler carbohydrates, generated by the hydrolysis of Spent Mushroom Substrate in the previous step. We pre-treated OM SMS, which did not give good butanol concentration without pre-treatment. After pre-treatment, the results were not significantly higher than when using OM SMS directly. One of the reasons may be that OM SMS doesn’t contain much xylan, as previously expected, and that the carbohydrates molecules consumed by strain S11 are the same when using OM SMS or hydrolyzed SMS. According to this remark, it can be concluded that SMS may not be a good substrate to be degraded by S11 strain. The cost of implementing a fermentation process using SMS as carbon source may be too high compared to the money that can be made by selling hydrogen or other VFAs, such as butyric acid. 71 4.5 Glucose concentration optimization This experiment is meant to evaluate which substrate concentration is optimal to grow S11 on glucose. The purpose is to find out the highest hydrogen and butanol concentrations, and at which glucose concentration these productions are achieved. Regarding the results of the previous experiment, the pH was controlled every day to pH=5.5, using H2SO4 or NaOH solutions. This was done in order to take full advantage of the previous results. The experiment conditions were the same as stated for the previous experiment: inoculation at 5% (v/v) of pure S11, every bottle was duplicated, and the fermentation was done anaerobically in the shaker 35°C, 150 rpm. The glucose concentrations tested in this experiment are the following: 2.5 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L and 60 g/L. The products concentrations were measured 13 days after inoculation. 72 40 C o n c e n t r a t i o n 35 30 25 CO2 20 N2 H2 15 10 ( m M 5 ) 0 2.5g/L 5g/L 10g/L 15g/L 20g/L 30g/L 60 g/L Figure 46 Gas composition of S11 pure culture growing on glucose at different concentrations at pH=5.5 Glucose 30 g/L show similar results to those obtained in the previous experiment when using exactly the same experiment conditions. This shows a good stability of S11 strain over time. As can be seen, when glucose is used as a carbon source, the highest hydrogen molar percentage is reached when glucose concentration is 60 g/L. However, it has to be noted that the molar yield of the experiment drops dramatically from 0.7 when using glucose 30 g/L, to 0.5 when using glucose 60 g/L. It is much more economically interesting to use glucose 30 g/L. 73 An interesting conclusion is that higher substrate concentration means higher hydrogen production, but doesn’t mean higher molar yield. There is a limit above which an increase of the substrate concentration doesn’t increase the hydrogen molar yield. These results have to be analysed on an industrial point of view. The main objective of our study is to generate value-added products for the mushroom farm. 4.6 Upscaling of the laboratory-scale experiments This part is meant to give a brief overview of what a farm-scale fermenter would be. The results of the experiments shown before this part are from experiments done on a laboratory-scale. We expect larger scale experiments to give the same results, but we don’t know if bigger microbial colonies would interact with each other, and disturb the fermentation process. We also don’t know how to deal with the larger gas production, produced by a larger bio-fermenter. 4.6.1 Protocol In a 4 Liter reactor, we are able to monitor the pH in real-time, which we cannot conduct in a 30mL bottle. pH is of paramount importance in our experiments, this is why it is crucial to control the pH as accurately and as regularly as possible. The fermenter used is a 4L glass-tank, with a magnetic stirrer. Three of the top openings were sealed using a rubber joint, and safely covered by a flexible plastic 74 paraffin film. The fourth opening is used to monitor the pH in real-time (by introducing a pH-meter). The fifth opening is linked, through a rubber joint, to a two-part flexible plastic tube (the two parts are linked with plastic paraffin film), whose other extremity goes to a 2L graduated plastic cylinder. This cylinder is reversed, filled with water, and stands using another cylinder to maintain the right pressure inside of the first cylinder. When gas is produced by the fermenter, the first cylinder goes up, pushed by the pressure difference between the gas and the water level inside of the cylinder. Figure 47 Bio-fermenter and gas tank at the initial step 75 Thus, the gas is continuously produced and stored in the cylinder. When the gas volume exceeds 2L, we use a 50mL syringe to release the gas from the cylinder. This syringe is inserted in the second part of the plastic tube which links the fermenter to the cylinder. Gas is pulled-out and precisely measured with the graduated 50mL syringe. Figure 48 Bio-fermenter and gas tank after 12 hours 76 We tried the scale-up experiment using SMS as the carbon source. However, there was no butanol produced: only Volatile Fatty Acids. Since our focus for this study is butanol, we used glucose as the carbon source in order to have both hydrogen and butanol productions: therefore we were able to analyze the results on a larger scale than when using the 30mL bottles. The methodology for the experiment is the following. We use 1.8L modified DCB1 medium, prepared as usual (see protocol in Chapter II). The strain used is S11 pure culture, inoculated at 5% (v/v). The results in the previous parts were best when using glucose 30 g/L as a carbon source in this experiment. pH is set to 5.5 after inoculation, and controlled as often as possible to pH=5.5, which is the best pH, as previously demonstrated. The fermenter was kept at air temperature (about 20°C), and shaked using a magnetic stirrer. We use the same experiment conditions as previous experiments, since we want to be able to compare the results on the laboratory-scale and on the pre-industrial scale. 4.6.2 Results As previously stated, the gas volume and composition were measured every day. Gas was continuously produced for 16 days. After 16 days, no gas was released anymore. So the total fermentation time is longer than when using the laboratory bottles. The reason to this fact is probably that the fermenter is much bigger than the laboratory bottles, so the bacteria growing in the fermenter have more difficulties to encounter the carbon source in the medium. Thus, it takes longer for 77 the bacteria to find and degrade the substrate in the fermenter. In order to make it on an industrial scale, an efficient stirring process should be implemented to improve the efficiency and shorten the time taken by the fermentation process. Following is the graph showing the cumulative gas production for 16 days. 18000 16000 14000 V o 12000 l u 10000 m e 8000 ( m L 6000 ) 4000 2000 0 1 2 4 6 9 16 Time (day) Figure 49 Total gas production of S11 pure culture growing on glucose 30 g/L on pre-industrial scale As can be seen, the gas production is still very high on the pre-industrial scale. The total cumulative gas production is 15,740 mL. The total volume of modified DCB1 is 60 times larger than when we use the laboratory bottles. Yet, the total gas production using the fermenter is 41.4 times the gas volume produced by the laboratory bottles. So the ratio is 0.69, meaning that there is a negative scaleeffect. If the mushroom farm were to implement our process, there would be a 78 slight difference in using a small volume or a high volume of medium: the scaleeffect is non-negligeable. We can even further guess than when using a larger fermenter, the total gas production will be even lower than when using a smaller volume. However, this is regarding the gas volume only. Bio-hydrogen and biobutanol can still be produced in large amounts, which is the focus of our study. Regarding the gas composition, following are the time-course results in the fermenter. 30 ( C o 25 n c 20 e n m 15 t M r a 10 t i 5 o n 0 H2 ) N2 CO2 2 6 9 16 Time (day) Figure 50 Gas composition of S11 pure culture in Fermenter As can be noted, the gas composition is very similar to the one found when using the small bottles. The molar percentage of hydrogen after 16 days is about 32%. Then, the biosolvent analysis was conducted at the end of the 16 days. The 79 fermentation process was complete (no gas production anymore), and a sample was taken from the fermenter to be analysed. 65.3 72.1 Acetic Acid Butyric Acid Butanol 112.7 Figure 51 Biosolvents production (mM) of S11 pure culture in Fermenter The results are again similar to those found when using laboratory bottles. The butanol concentration here is 4.84 g/L, which is slightly lower than when using the laboratory bottles. However, butanol is still produced in reasonable quantities, and this fact could be exploited on an industrial scale project. Thus, using the same process and parameters (pH=5.5, substrate concentration set to 30 g/L) on a pre-industrial scale gives similar results to those obtained when using laboratory bottles. Hydrogen and butanol are generated by S11 strain in almost the same concentrations, but the total gas volume is about 30% lower. 80 Nonetheless, it has to be noted that the process is slower than when doing the experiment on a laboratory scale. If this process were to be implemented on an industrial scale, a very efficient stirring process should be built. Moreover, a goodquality fermenter should be built, with perfectly fitted tubes to continuously conduct gas out of the fermenter to a tank, cylinder, or membrane process; and with all the safety precautions due to handling hydrogen in non-negligible quantities. 81 CHAPTER 6. Conclusion 6.1 Major findings With reference to the problem statement and the aims set out earlier in this report, the objectives of this study have been met. Listed below are the central findings of this study. First, we disclose a fermentative pathway generating value-added products, using bacteria isolated from Spent Mushroom Substrate. This process generates high concentrations of bio-hydrogen and bio-butanol from glucose. The methodology has been fully detailed earlier. It is an easily-implementable and reliable way for mushroom farms to produce on-site bio-energy. Second, the fermentative bacteria were isolated from Spent Mushroom Substrate and several kinds of carbon sources were tested. The strain isolated and used for the major part of the experiments in this paper is named S11. A full DNA analysis showed that this strain is a Clostridium sp. Third, we identified two main kinds of SMS, which were degraded by strain S11: the one used to harvest Oyster Mushroom (OMN) and the one used to harvest Abalone Mushroom (A). Other kinds of SMS were tested, but did not show could results when being degraded by S11. 82 Fourth, we optimized the experiment conditions to obtain the highest amounts of bio-hydrogen and bio-butanol. We found out that the optimal pH for the fermentation process is 5.5. The optimal substrate concentration is 30 g/L when using glucose as carbon source. This concentration does not give the highest amounts of value-added products, but is the most economically relevant, because the yield of the experiments drop when glucose concentration is higher. After a study of the fermentation process of S11 on two different kinds of Spent Mushroom Substrate, the conclusion is that butyric acid is produced in relatively high concentration. This value-added product could be sold by mushroom farms, who could produce butyric acid on-site. Fifth, we also implemented a pre-industrial scale experiment, using glucose as carbon source, which showed good products output. There seems to be a slight negative scale-effect: further study should be done in order to improve this weak point. 6.2 Recommendations and future studies The outlook of this study seems promising. Obviously, the most efficient means to prevent further waste generation of Spent Mushroom Subsrate lies in a better SMS management. Farmers should try to reduce their SMS use. They tend to replace their Spent Mushroom Substrate too often in order to increase the productivity of their mushroom crops, which inevitably produces a very high quantity of waste. They should also try to reuse it as a compost material, which is the simplest way, 83 today, of handling their SMS. Our process is a cost-effective and energy-efficient way to generate value-added products using bacteria isolated from Spent Mushroom Substrate. It is also worthwhile to pay attention to the development and the extension of the knowledge we currently possess in the field of genetic engineering of microbes. With the combination of an in depth understanding of biochemistry and genetics, and the applications of recombinant DNA techniques, it is possible to characterize the appropriate genes and transfer them to construct engineered strains, derived from our S11 strain, with enhanced capability for degradation of different substrates. The combination of an optimized fermentation process with a genetically engineered S11 strain would probably lead to very high outputs of value-added products. While we acknowledge and appreciate the contributions and works that have been carried out in this field, there is so much more that needs to be accomplished in this direction. Further study should be done in order to assess the total investments needed to implement our on-site solution. 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Arch. Microbiol. 153:267-271, 1990 89 [49] Cole, Fathepure, Tiedje. Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced in Desulfomonile tiedje DCB-1. Biodegradation 6:167-172, 1995 [50] Dolfing, Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-1. Arch. Microbiol. 153:264-266, 1990 90 [...]... low-carbon technologies We emphasize the need to generate value-added by- products from clean sources, such as bacterial fermentation We will highlight the importance of hydrogen and butanol, both produced by either a mixed culture of bacteria or single-strain colony, isolated from Spent Mushroom Substrate 1.2.1 The potential of hydrogen Hydrogen is considered to be one of the most promising fuels of the... Table 1 Components of Spent Mushroom Substrate Source: [12] 3 In this study, we use the Spent Mushroom Substrate from an organic mushroom farm located in Johor, Malaysia The SMS was used to harvest several species of mushroom, mainly Pleurotus Ostreatus (OM, Oyster Mushroom) , Lentinula Edodes (LE, Shiitake), and Pleurotus cystidiosus (A, Abalone Mushroom) Oyster mushroom is a mushroom species commonly... compost material is called spent , and it is then replaced by fresh compost Despite the evident benefits of mushrooms, the exponential increase in their consumption worldwide is also generating a high volume of spent mushroom substrate (SMS) Spent Mushroom Substrate is discarded and treated as waste It has been reported that about 5 kg of substrate are needed to produce 1 kg of mushroom [2], and about... one mushroom culture cycle is about 4 months Then the anaerobic plastic bags were brought to our laboratory one month after the harvest of the mushroom Spent Mushroom Substrate was made of different materials, depending on the mushroom to be grown on it For OM, SMS was made of oak tree sawdust, from China For A, SMS was made of rubber tree sawdust, from Malaysia This mushroom farm is an organic mushroom. .. promote butanol fuel, claim that "biobutanol can be blended up to 10%v/v in European gasoline and 11.5%v/v in US gasoline" [31] According to analysis done by the National Renewable Energy Lab [32], most of the advantages of butanol come from its properties as a fuel, not from current production technology Traditionally, low yields - in the 15 to 25 percent range have plagued butanol production from bacterial... searched for Hydrogen gas production from renewable resources such as biomass, and carbohydrate rich waste materials by bioprocesses, offers distinct advantages over energy intensive methods used Major drawbacks in biohydrogen production are low yields and productivities requiring large reactor volumes and long residence times [23] As mentioned beforehand, nowadays, hydrogen is usually produced by thermochemical... problems in bio- photolysis of water [24] Fermentative hydrogen gas production from carbohydrates is a much faster process than bio- photolysis, with volatile fatty acids (VFAs) and H2 gas formation [25] However VFAs need to be fermented for further H2 gas production [26] Major mechanisms for bio- hydrogen production by fermentation have been 10 elucidated However, development of an effective bio- hydrogen. ..List of tables Table 1 Components of Spent Mushroom Substrate Source: [12] ……………….3 Table 2 PCR conditions ………………………………………………………… 25 Table 3 Hydrogen yield of S4, S11 and ESE1 on Glucose 30 g/L ……………….49 Table 4 Standards composition ………………………………………………… 67 x List of symbols SMS – Spent Mushroom Substrate OM – Oyster Mushroom LE – Lentinula Edodes A – Abalone Mushroom GC – Gas Chromatography FID –... and bio- conversion processes Among them, bioconversion process is a promising method for two reasons: utilization of renewable resources, and usually operated at ambient temperature and atmospheric pressure Main bio- hydrogen production processes are direct or indirect bio- photolysis, dark and photo-fermentations Bio- photolysis of water under sunlight is considered as the cleanest approach for bio- hydrogen. .. chemicals Source: [19] Hydrogen gas is also a clean fuel with no COx, SOx and NOx emissions from its combustion Besides, hydrogen is an important energy carrier and can be used in fuel cells for generation of electricity [20] However, hydrogen gas is not readily available in nature like fossil fuels and natural gas, but can be produced from renewable materials such as biomass and water [21] Hydrogen gas production ... the mushroom industry The main objective of our study is to isolate and cultivate bacteria from this Spent Mushroom Substrate An anaerobic fermentation process producing bio- hydrogen and bio- butanol. .. then Spent Mushroom Substrate as carbon sources for the micro-organisms Few previous studies were done on this topic In this study, three strains of bacteria were isolated from Spent Mushroom Substrate, ... directly in a mushroom farm v One strain was particularly good in generating bio- hydrogen and bio- butanol, and was named S11 However, most of Spent Mushroom Substrates were not easily degraded by S11