A Sterilization of Equipment
	All equipments will be washed thoroughly with liquid detergent and water. They will then be oven dried at 100oC for over 30min. With the use of paper, the equipments will be wrapped. They will then be autoclaved at 15 psi and 120oC for 30min. After the autoclave will have been turned off, the glassware will be allowed to be cooled before they will be taken out of the autoclave.

B Collection of Waste Samples
	Samples will be collected from confectionery wastes, cow fecal matter and wastewater. These samples are ideal for the existence of Escherichia coli since they enclose sugars and carbohydrates necessary for the hydrogen-producing metabolism of the said bacteria. All wastes will be collected on the same day. Sterile, non-toxic, glass containers with a leak-proof lid will be used in the waste collection. The containers will be capable of holding 250ml. All companies pertaining to the waste samples will be informed of the drawing together of samples before the actual collection.
	B.1 Confectionery Waste
	Confectionery wastes will be obtained from the factory of ¬¬_________. Samples will be obtained from the waste accumulated from the manufacturing of their sweet delights. It will be expected that the samples will contain a combination of caramel, sugar, bread, etc. The waste will be collected and put in 250 ml containers while wearing latex gloves so as to maximize the sterility of the procedure. It will be made sure that at least 2-inches of headspace are kept in the glass containers. The ample 2-inch headspace will be provided to facilitate the mixing of samples by shaking prior to the isolation. Immediately, following the sample collection, the sample container’s lid will be tightened.
	B.2 Cow Fecal Matter
	The manure of cow (Bos Taurus) will be used in the study. The cow will be from the _________. It is in the feces of mammals wherein E. coli is usually found. Fecal material will be obtained by palpating the rectum of the cow and collecting 10g of feces using a clean, plastic palpation glove. The material will then be placed in a Ziploc bag and stored in the cooler before transportation.
	B.3 Wastewater
	In the wastewater treatment facility of the _________, wastewater will be collected. Wastewater samples will be collected using the Hand-Dip (Grab) Method with a sterile 250ml glass container after the collection. The base of the sample bottle will be grabbed with one hand and the bottle cap will be removed. Then the bottle will be inverted and it will be plunged into the wastewater about 15cm (6 inches), and then the bottle will be tipped mouth up toward the water surface. Samples will then be chilled in the cooler. Similar to what was done with the confectionery wastes, at least 2 inches of headspace will be kept and the containers will be sealed immediately after putting the sample. Latex gloves will also be worn in doing the procedure.
	B.4 Transportation
	All samples will have already been cushioned inside the cooler filled with ice. As soon as all waste samples have been collected, they will be rushed to the laboratory between the time of collection and initiation of experiments, which would never exceed 24 hours. 
	B.5 Storage
	All the waste samples will have already been put in the glass containers and stored in stored in an icebox filled with ices so as to maintain the presences of Escherichia coli. They will then be rushed to the laboratory and stored in the refrigerator. All the samples will be kept at ~4oC.

C Isolation of Escherichia coli in the Waste Samples
	C.1 Preparation of EMB (Eosin-Methylene Blue) Plates
Eighteen grams of dehydrated culture media will be suspended in 500ml of contained distilled water in a media bottle. The suspension will be heated to boiling and mixed until the culture media will be dissolved completely. The agar will then be sterilized in the autoclave at 121oC and 15 psi for 15 min. The agar will then be cooled to 45-50oC and will be dispensed in Petri dishes.
	


C.2 Dilution of Waste Samples for Standard Plate Count
	There is a need to dilute the waste samples so as to limit the growth of the bacteria. One method of diluting samples is the Pour Plate Method requires use of 1 ml, 0.1 ml, and 0.01 ml or 0.0 01 ml of the samples. The difficulty of measuring and working with the two smaller volumes, 0.01 and 0.001 ml, require the use of sample dilutions. These dilutions are prepared by pipetting 1 ml/ 1kg of undiluted sample into 99 ml of distilled water. Diluting the sample allows 1 ml of diluted sample to be used instead of 0.01 ml of undiluted sample. 
See Figure. 1 Sample Dilution below
Fig. 1. Sample Dilution with Pour Plate Method
	C. 3 Isolation Procedure
	E. coli from the waste sample will now be isolated with three replicates from each source. A thin layer of the diluted waste materials will be roughly poured unto the prepared EMB plates and spread all over using a stirring rod. Plates will then be labeled according to the waste sample inside and the date of collection. The plates will be incubated at 37oC for 24 hours.
	To obtain pure cultures of E. coli (since the agar plates now contain a mixture of isolated organisms), a single E. coli colony from each plate will be streaked on other EMB plates. E. coli colonies can be easily identified by its characteristic purple color and metallic green sheen. The needle end of a sterile loop will be used to touch well isolated E. coli colonies in the plates. The needle will then be streaked across 1/3rd of the previously prepared EMB plates. They will then be incubated aerobically at 37oC and 250 rpm. Plates will then be labeled with the waste sample in which the E. coli was isolated, the date of incubation and the replicate number.
	C.4 Determination of Bacteria Concentration
	Bacteria concentration will be recorded using the McFarland Standards. McFarland Standards are generally labeled 0.5 through 10 and filled with suspensions of Barium salts. The standards will be made in the lab by preparing a 1% solution of anhydrous BaCl2 and 1% solution of H2SO4 – mixed them in the proportions listed in Table 1. They will be stored in the dark, in a tightly sealed container at 20-25oC and should be stable for approximately 6 months.
Table 1. Approximate E. coli concentration on McFarland Scale
	Each tube approximates the turbidity of bacterial  solutions corresponding to the McFarland Scale number. Thus tube 7 represents the turbidity of bacteria at a concentration of 2.1 x 109/ml. The rough population of the culture can be quickly determined; its turbidity can be visually compared to a set of McFarland Standards in Table 2 (below). If its turbidity falls between tubes 7 and 8, then the number of bacteria/ml will be between 2.1 and 2.4 billion per ml. The advantage of these standards is that no incubation time or equipment is needed to estimate bacterial numbers. The concentration of E. coli will then be recorded. The concentration of         E. coli will then be recorded for each replicate from each source.
Table 2. The McFarland Scale is a scale numbered from 1 to 10 which represents specific concentrations of bacteria/ml. It is designed o be used for estimating concentrations of gram negative bacteria such as E.coli
D. Hydrogen Low-Partial Pressure Assay
	D.1 Preparation of LB (Luria-Bertani) agar
	In a graduated cylinder, 250 ml of distilled water will be added. Premixed LB agar powder will be weighed for 20g using an analytical balance. If a premixed LB agar powder is not available, 5.0g tryptone, 2.5g yeast extract, 5.0g NaCl and 7.5g agar will be blended. The powder will then be brought and mixed with the distilled water well in the graduated cylinder. The cylinder will then be added with distilled water up until a total volume of 500ml will be achieved. 
	With a pH-meter or pH paper available, a small aliquot of the solution will be taken to be checked for the pH. If it will fall well below pH 7, additional distilled water will be added so that the pH will be adjusted to 7-7.4. This is done so that the LB agar will not be too acidic for the growth of bacteria.
	The solution will then be transferred to a 1L Erlenmeyer flask. The Flask will then be put on a stirring hot plate to boil the LB solution for 1 min while stirring. The heated solution will be transferred to a 1L glass jar and labeled. The opening of the flask will be covered with tin foil and will be placed in an autoclavable plastic or metal bin with some water in the bottom (~1 cm deep).
	It will then be autoclaved at a liquid setting for 20min 15 psi. The LB agar will then be cooled to ~55oC. After autoclaving, the lask will be gently swirled while holding it oven mitts. This action is necessary to insure even distribution of the agar in the media; else it often remains denser near the bottom.
	 Each plate will be left to cool for 20 min (until it is solid). They will be flipped so as to avoid condensation on the agar. All the plates will then be stored back in the plastic bags and into the refrigerator kept at 4oC.
	D.2 Flasks for Hydrogen Production
The following procedures are adopted from Maeda and others (2008)
	The 250ml flasks will be filled with 75ml of complex-glucose media. Overnight cultures will then be transferred to the flasks with tubes. Flasks will be closed with rubber stoppers that include the tubes will be sparged with nitrogen for 5min. Flasks will be sealed and incubated anaerobically at 37oC for 6h. This hydrogen production is done under anaerobic conditions. To duplicate this experiment, a tank of nitrogen will be needed. The nitrogen gas will be sparged through the culture medium to keep oxygen out. If E. coli has access to oxygen, they will use the oxygen to ferment the sugar to form carbon dioxide, so it is important to keep oxygen away. Thirty milliliters of complex-media without glucose will be sparged with nitrogen for 10min using flasks connected with tubes. Media with 1M glucose will be sparged with nitrogen for 5min (7 to 10ml) using sealed 60ml glass vials with stirrers. Glass vials will be sealed tightly with a Teflon cap and sparged for 2min with nitrogen.
		D.2.1 Sparging
		In sparging glass vials with nitrogen gas, a needle will be connected with the input of nitrogen gas, and another needle will be for the output of gas. After sparging with nitrogen gas, the needle for the output will be closed using the head cap. Then, after 30sec, the vials will be ensured that it did not leak gas, gas flow would be expected to stop.
	D.3 E. coli Inoculation with Glucose-Rich Medium
	A glovebox will be introduced with the flasks with cultures, and a 250ml centrifuge tube per culture. The glovebox will then be purged with nitrogen. Inside the glovebox, the cultures will be transferres to the 250ml centrifuge tubes. Tubes will then be removed from the box and centrifuged at 7000 rpm, 4oC for 5min. Inside the glovebox, the supernatants will be poured and the cell pellets resuspended in the 30ml of previously sparged complex medium without glucose. Eighteen milliliters cell suspensions will be transferred to 60ml glass vials using a syringe. With a syringe, 2ml of sparged 1 M of glucose will be added to the glass vials. The vials will then be removed from the glovebox and is ready for the set-up according to Fig 2 (next page), and will be incubated at 37oC.
 
Fig.2 Hydrogen Low Partial Pressure Assay
		D.4 Containers with 1M NaOH
		One Molarity NaOH is a solution containing 1 mole of sodium hydroxide dissolved in a liter of water. It is usually made by dissolving sodium hydroxide pellets in distilled water, but it can be purchased as the solution from a scientific supply company. To make a solution, the molecular weight of sodium, oxygen, and hydrogen would be added up to obtain the grams/mole which would be 40g/mol. Then in one liter of water, 40g of NaOH will be dissolved. The solution will then be transferred to the vials filling them up with 60ml each. Vials will then be closed with a rubber stopper connected with two tubes (The first tube will be submerged in the NaOH solution while the second tube will be suspended in the headspace.
		The purpose of the NaOH in the experiment is to remove carbon dioxide from the hydrogen gas produced by E.coli. If this step will not be included then the gas collected would be a combination of carbon dioxide and hydrogen gas. Produced biogases will be allowed to leave the headspaces of the vials through a needle in the septum via tubing that will direct the gas through the vials filled with NaOH. Carbon dioxide will be removed in the biogas due to its reaction with NaOH. The isolated hydrogen gas will be directed again by the tubing into an inverted graduated cylinder filled with water. Fresh NaOH will be needed for use in each experiment. 
E. Quantification of Hydrogen Production of E.coli
E.1 Measuring the Volume of Hydrogen
The set-up shown in Fig. 2 of the reference shows a graduated that is initially filled with water and inverted in a beaker of water. Tubing from the culture vessel on the stir plate ends in the graduated cylinder, so any gas produced from the culture will be released in the cylinder and the water will gradually be replaced by hydrogen gas, and the volume of gas produced can be measured. After each measurement, the graduated cylinder used will be replaced by new cylinder.
The only possible path for hydrogen gas to escape is through the tubing, which ends in the glass cylinder (see Fig.2). The gas will rise in the water and displace it, and since the gas will be in a graduated cylinder, the volume can be measured. This set-up allows capture of all the hydrogen gas. Hydrogen gas volume of each replicate from each source will be taken. Volume of the hydrogen will be measured in grams per milliliter (g/ml). All measurements will be recorded and written in a spreadsheet.
	E.2 Confirming Hydrogen Production
	Hydrogen gas is clear; it looks like air. It will be verified that the gas is hydrogen by inverting the cylinder and quickly lighting it with a lighter or a match with a long handle while wearing safety glasses and non-flammable clothing. Hydrogen gas is flammable and a flash of flames will be seen as the hydrogen burns.
F. Assessment and Comparison of the Hydrogen Production by E. coli from different sources
	A line graph will be formulated using the software Microsoft Excel®. The graphs will have the hydrogen production performance of each strain and its replicates in the y-axis and the time of incubation in the x-axis. The optimum producer will then be identified and recorded.
G. Handling and Disposal
H. Safety Procedures