Industrial Microbiology and Biotechnology Final

What are the two ways to measure the concentration of sucrose over the course of a fermentation?

Hydrometer Refractometer

What are two reasons for which wine is filtered?

Clarity Stability

What is pitch?

The starter culture for fermentation.

Which has a higher osmolarity: 1 M NaCl or 1 M sucrose? Where does 1 M glucose fall in this ranking?

1 M NaCl has a higher osmolarity given that the sodium and the chloride dissociate (thus, causing them both to contribute to osmolarity). Glucose and sucrose have nearly identical osmolarities given that neither of them dissociate.

Why is it necessary to sparge in oxygen at the beginning of a fermentation run with yeast? Why do you not need to do this with zymomonas?

1) This allows them to generate a lot of ATP that can be used for initial growth to reach an intended biomass 2) The synthesis of oleic acid and ergosterol (necessary for EtOH tolerance) requires oxygen. Zymomonas, however, can synthesize the equivalents of these (cis-vaccenic acid and hopanoids) without oxygen.

What is a refractometer?

A device used to measure the concentration of sucrose that reads the refractive index of the solution relative to water (given that sucrose has a different refractive index compared to water).

What is an appellation?

A geographical area that is designated for growing certain types of grapes that are used for specific types of wine (needs to be made in this area).

What is Gruit?

A mixture of various herbs and spices that was historically thrown into beer to try and get rid of "the bad stuff" in beer that would result in a bad brew (microbes).

What is malolactic fermentation?

A pathway performed by some lactic acid bacteria such as Oenococcus oeni in which malic acid (a dicarboxylic acid) is converted to lactic acid and CO2.

What is bottle conditioning?

A process of carbonation used mostly by home brewers. This is done by adding a controlled amount of yeast to generate carbon dioxide.

How is diacetyl produced during malolactic fermentation?

A side activity of Oenococcus oeni is to take citric acid and convert it to diacetyl through an entirely separate pathway from malolactic fermentation.

What is a late harvest wine?

A sweet wine made from grapes infected with the fungi, Botrytis cinerea (Noble rot).

What kind of flavour would you obtain by leaving behind more sugar during the mashing step (converting less to glucose)?

A sweeter/lighter beer

What is a fino sherry?

A type of Sherry that is aged with a lower concentration of alcohol to allow for a thin film of microbes to grow on the sherry (Flor), resulting in interesting flavours.

What is port?

A type of fortified wine produced in Portugal's upper Douro Valley. These are sweet, highly alcoholic wines that are made by stopping fermentation early (because they hadn't discovered sugar cane yet when this was first made) by dumping in brandy to preserve many of the residual sugars from the grapes.

What two compounds do you want to avoid the production of via yeast in wine making?

Acrylamide Ethyl Carbamate

What are the two types of yeast? What distinguishes them from one another?

Ale yeast (Saccharomyces cerevisae) - Top fermenting yeast that accumulate at the top of a brew. Lager yeast (Saccharomyces uvarum) - Bottom fermenting yeast that accumulate at the bottom of a brew

What are the two types of starch and what differentiates them?

Amylose (Unbranched α1,4 linkage) Amylopectin (Branched α1,4 linkage and α1,6 linkage)

What two things make late harvest wines tricky to ferment?

Anti fungal compounds secreted by the Botrytis (inhibits yeast growth) High sugar concentration imposes high osmotic pressure on the yeast.

Why do hops provide an edge in beer brewing?

At the end of the mashing process, there is about 10% sugar in the wort, which isn't a high enough sugar concentration to inhibit other non-yeast microbes from growing. Thus, hops help inhibit their growth.

During which step of beer making can a beer be made "darker"? How?

Beer can be made darker by letting the malting stage run for longer than usual, allowing for more conversion of starch to glucose.

What is unclarified beer?

Beer that has not been properly fined and is generally unhopped. These beers contained suspended solids (Ie. proteins and some microbes).

What is often termed as the initiator of the biotechnology revolution? Where did this begin?

Beer-making. The beer-making business began in Mesopotamia (starting with Egypt).

What is the reaction for the yeast fermentation in beer and wine?

C6H12O6 -> 2 C2H5OH + 2 CO2

What are the two major fining/clarifying agents?

Chitosan: de-acetylated chitin that is generated from chitin via acid treatment. This is a by-product of the shellfish industry and is cheap to produce en masse Diatomaceous earth: Beds of dead diatoms that settle into the sand that are collected and treated with nitric acid (to remove organics).

Which has more calories per gram: glucose or ethanol?

Consider the following reaction: C6H12O6 -> 2 C2H5OH + 2 CO2 + 2 ATP Very little is energy is lost when ethanol is produced, but a considerable portion of the mass is lost. Therefore, ethanol has a higher caloric density.

What are the three types of substrates for bioethanol production? What are the components of each?

Corn (comprised of starch, which is converted to glucose) Sugar cane (comprised of sucrose, which is converted to glucose and fructose) Cellulosic materials (Ie. wood, grasses, corn stove; comprised of lignin, cellulose, and hemicellulose)); note hemicellulose is really the own one of these components that can be a good carbon source (it is comprised of mostly pentoses)

What are diastases? What is diastase activity?

Diastases: A general term referring to any enzyme involved in breaking down starch in some form. Diastase activity: A grain's ability to undergo malting.

How do yeast produce glycerol?

During fermentation, an intermediate product, Dihydroxyacetone phosphate (DHAP), can undergo a side reaction to form glycerol (dephosphorylated DHAP)

When are hops generally added during the beer-making process?

During the wort boiling step.

What are three types of yeast that are common in natural fermentations?

Kluyveromyces Torulaspora Zygosaccharomyces

What are pectinases?

Enzymes that help break down the skins of grapes to release the pigments.

How is ethyl carbamate produced? Why is it dangerous?

Ethyl carbamate is produced from the spontaneous conversion of ethanol and urea to Ethyl carbamate. While this compound isn't dangerous in itself, it can eventually form vinyl carbamate, which can form vinyl carbamate epoxide. Epoxides are dangerous because they can interact with free nucleotides and genomic DNA (thus, a carcinogen/mutagen).

Why is it better to add sucrose into a fermentation mixture as opposed to glucose?

Given that 1 M glucose and 1 M sucrose have the same osmolarity, you can add more sugar by adding sucrose (disaccharide) as opposed to glucose without raising the osmolarity.

What are spent grains? What are they used for?

Grains left behind after the lautering step. They are comprised of protein, fat, fibre, and any remaining starch. These are generally used for cattle feed.

What are lactic acid bacteria? How were they prevented from ruining a brew?

Gram-positive bacteria that grow well in environments with high sugar content. They can cause a beer to taste sour. The addition of hops to the worth helped inhibit their growth.

What types of microbes do hops inhibit?

Gram-positive bacteria/lactic acid bacteria growth is inhibited by hops

What are four types of biogenic amines that can be produced during malolactic fermentation and from what?

Histidine >> histamine Tyrosine >> tyramine Ornithine >> Putrescine Lysine >> Cadaverine

What are the two ways of preventing glycerol from being made during yeast fermentation?

If the glucose concentration is low, glycerol won't be made. Thus, this can be achieved in two ways: 1) Add glucose gradually 2) Simultaneous Saccharificatio - you add a calculated amount of enzymes to produce glucose at a constant, low concentration OR you add Aspergillus oryzae to break down starch into glucose gradually (not really used industrially, but this is used for making Sake.

Why do beer brewers and alcohol producers often start "fermentation" by bubbling oxygen into the tank?

If you add oxygen, the yeast will start growing rapidly (this might be faster than anaerobic growth). However, they produce significantly more ATP during aerobic respiration as opposed to fermentation (36 ATP produced). Thus they can get yeast to a suitable population density without consuming too much sugar in the process (more efficient).

Why do you not want to obtain 100% conversion of starch to glucose?

If you leave a little bit of oligos around - referred to as dextrins (chains of 4-7 glucose monomers) - they add a sweet flavour to the brew, given that yeast can't ferment them, so they stick around in the final brew.

What are three advantages of malolactic fermentation?

It makes the wine taste less sour It stabilizes the wine (good for long storage) It imparts a buttery flavour via the production of diacetyl

What is the function of α-amylase?

It rapidly hydrolyzes starch (both amylose and amylopectin at the α1,4 linkage).

What is the source of enzymes for the mashing process?

Malted grain

What are the 8 steps of the beer-making process (in order)?

Malting Grinding Mashing Lautering Wort-boil Fermentation Clarification Carbonation/bottling

In what forms do brewers used hops

Pelletized hops (most common) Hop extract oil (doesn't aid in protein precipitation) Compressed hops (Buddweiser does this. Probably why it tastes terrible)

What two things do you usually add at the end of wine fermentation to stop it?

Potassium Metabisulfite (to kill off bacteria/molds) Potassium Sorbate (to kill off the yeast/fungi)

What is the function of sorbic acid and in what form is it added?

Potassium sorbate is added to wine after the fermentation (or near the end of it) to inhibit the growth of yeast and other fungi. It does so by collapsing the pH gradient of the membrane.

How do bacteria get energy from malolactic fermentation?

The bacteria takes in malic acid via an antiporter that simultaneously spits out protonated lactic acid (which dissociates afterwards), generating protein motive force.

What are lectins? Which one is involved in flocculation? Why is it ideal to have a strain of yeast that constitutively expresses this particular lectin?

Proteins that bind to sugars. Flo1, found on the surface of some yeasts, is involved in the process of flocculation. It preferentially binds to mannens (Also found on the surfaces of yeast), causing yeast to accumulate/clump together. It is ideal to have a strain of yeast that constitutively expresses flo1 given that this binding is too weak to allow for accumulation during fermentation (when the brew is mixed), but will take effect after everything settles.

What are 6 characteristics of an ideal yeast strain for winemaking?

Resistance to high sugar concentrations Insensitivity to sulfur dioxide (so you can get a wide margin between what kills other contaminants and what kills yeast) High ethanol tolerance (some yeast can take up to 17-18%) Resistance to tannens (which have some anti-microbial properties). Good flocculation Good flavour profile

What are the three types of organisms used for making bio-ethanol?

Saccharomyces yeast Zymomonas mobilis Clostridium thermocellum

What is an alternative to using lactic acid bacteria for malolactic fermentation?

Scientists engineered a wine yeast strain (MLO1) to perform malo-ethanolic fermentation (Reducing malate without the use of lactic acid bacteria. This was done by cloning in mae1 (a malate permease) and mae2 (catalyzes the conversion of malate to pyruvate, which is converted to ethanol). This avoids the disadvantages of malolactic fermentation, but you don't get diacetyl production (buttery flavour).

How is Sherry made?

Sherry, a fortified wine produced in Jerez, Spain, is made via the "solera" system, in which white wine is poured into the top of a series of stacked, interconnected barrels (with each barrel only being filled about half way to two thirds). Thus, the wine trickles down to the bottom, where the older wine is withdrawn from. This is done to expose the wine to oxygen to induce oxidation.

What are the four ways of sealing a wine?

Standard corks Agglomerate corks (tiny pieces of chopped cork bound together by glue) Synthetic closure The screwcap

What are the four major kinds of acids found in wine?

Tartaric Malic Lactic Citric

What are the three compounds that were used to prevent gasoline from burning too readily and to oxygenate the gasoline?

Tetraethyl lead (the leftover lead acts as a great lubricant for the engine, but is bad for humans) MTBE (outlawed because it penetrated ground water) Ethanol

When performing malolactic fermentation, why do you not want to inoculate the wine with Oenococcus oeni too early?

The Oenococcus oeni will convert glucose to lactic acid instead of ethanol, reducing the yield of ethanol.

What grain was beer initially produced from in Babylon? What caused the shift?

The Sumerians and Akkadians first began commercially producing beer from Emmer wheat. However, Emmer wheat is a salt-sensitive grain. Given that they grew this wheat in the irrigated "fertile crescent", the land was susceptible to an influx of melted snow that evaporated during the summer, depositing salt in the soil (salting up). Thus, they shifted to Barley, which is less salt-sensitive.

What are 7 regulations in place for Ice wine producers?

The grapes must be naturally frozen The wine "must" must have a brix of 35 or higher Must have a residual sugar of 125 g/L at the end of the fermentation No fortification (The alcohol must be derived exclusively from the grapes) Must be a registered grower/brewer of iced wine The harvest must happen after November 16th You must harvest and crush at a temperature of -8°C or lower.

What are two reasons for which wine making was historically more reliable than beer making?

The high sugar content in wine (from the grapes) inhibits the growth of many other contaminating microbes. The higher ethanol content is toxic to other contaminating microbes

What is the main goal of the mashing step in beer making? How is this done?

The main goal of mashing is to facilitate the final conversion of most of the remaining starch to glucose (but not all). This is done by taking the dried, ground-grain, hydrating it again, then heating it back up to 50-60°C, after which the enzymes are denatured at around 65-70°C.

What is humulone?

The major alpha acid in hops. It gets converted to isohumulone during the wort-boiling process, which is more bitter.

What is fortification? How was this historically done?

The process of adding extra ethanol to the wine under the premise that it should inhibit most bacterial/yeast growth (good for preservation).

What is malting? What is the main goal of it?

The process of convincing the plant to break down starch to produce glucose (but not for growth). The main goal of this process is to wake up the diastases in the grain being malted (generally barley) by heating the grain to 50-60°C.

What is a mashing profile?

The specific procedure a brewer follows during the mashing process. This varies quite a bit depending on the intended brew.

Why was beer making not a reliable process in the middle ages?

There wasn't always enough sugar in the wort for the yeast to grow (used up by other microbes).

What are Oenococcus oeni and what are they used for?

These are lactic acid bacteria (gram positive) that are aerotolerant (grow in the presence of oxygen, but don't use it). They can't convert glucose to CO2. Rather, they convert it to lactic acid (instead of ethanol). They perform malolactic fermentation and are thus used to get rid of any malate in the wine.

What are chardonay grapes chosen for?

They are chosen specifically for the amount of lactic acid fermentation that will occur (imparting its buttery flavour).

Where are the enzymes that break down starch during the malting and mashing process produced? What are they naturally used for?

They are produced in the grain being malted (generally barley). These are ordinarily used to germinate from the seed, given that the plant needs to produce enzymes to break down the starch to allow growth.

How can yeast strains be engineered to avoid ethyl carbamate production?

They can be engineered to constitutively express Dur1,2, allowing for continuous conversion of Urea to NH3.

What are the four functions of acids in wine?

They preserve it, enliven it, shape the flavours and prolong the aftertaste.

How do yeast tolerate high sugar concentrations?

They produce glycerol, which is a compatible solute (a compound that a cell produces to prevent water loss under high osmotic pressure given that it balances osmolarity).

What are two characteristics of good grapes for making ice wine?

They should we well-rounded marbles They should not be affected by fungi like botrytis.

What effect does potassium metabisulfite achieve in winemaking?

This can be used to kill a certain fraction of the microbes in solution due to the following dissociation that occurs: K2S2O5 -> K2O + 2 SO2. The sulphur dioxide can be used to kill microbes (as yeast are relatively insensitive to this). This avoids the addition of acetic acid or other bad flavours from unwanted microbes/molds.

What are two disadvantages of malolactic fermentation?

This is difficult to control, given that malolactic fermentation is a pretty bad source of energy for Oenococcus oeni and if you add it to early, they'll just operate under their normal pathway and metabolize glucose. They also can produce biogenic amines (Ptomaines), which might be the reason people get headaches from drinking red wines.

Why is Sherry fortified during its agin process?

This is to avoid microbial growth during its aging process that might otherwise spoil it.

What is the purpose of fining?

This step in the beer-making process helps settle out the remaining bits of grain and yeast in the finish.

What is the main goal of the grinding step in beer making?

To grind all of the dried malted grain just well enough to expose most of the starch to the water while not grinding it too fine.

What are the three purposes of metabisulfite in wine making?

To kill off molds, bacteria and unwanted yeasts before natural fermentation. To kill off all natural flora to control the inoculum. After fermentation, it can be used again to kill bacteria, function as an oxidant (to protect colour and flavour) and to stop yeast from growing (when used with sorbate).

What is the main goal of the lautering step?

To obtain the wort by filtering the liquid in the mash through the natural filter produced by the grain husks.

What are the four reasons for which the Wort is boiled?

To precipitate proteins (they make the beer cloudy) To extract the hop flavor To kill off any microbes (mainly bacteria, but also other yeasts) To stop all diastase activity

What are the three functions of hops?

To suppress bacterial growth (mainly gram-positive bacteria) To aid in clarification (helps to precipitate proteins) To result in desirable flavours in the final brew

What are three ways of preventing cork taint?

Trim off the bark that's closest to the ground (don't use it) Treat the bag of corks with sulfur dioxide gas Extract the TCA with ethanol/kill microbes

Why is it required to oxygenate gasoline? How much ethanol or MTBE would you need to add to have 2.7% oxygen (as required by the clean air act?

Unburned gasoline/carbon monoxide can create smog. This can be solved by introducing more oxygen into the gasoline. To reach a 2.7% oxygen, you could add 7.5% ethanol or 15% MTBE.

How was Brandy originally made?

Via the distillation of wine, given that ethanol evaporates first, allowing for concentrated ethanol to be obtained.

Why is wheat not ideal for the grinding step in beer making?

Wheat doesn't have as hard a kernel as barley. Thus, it can easily plug up the filter during the lautering step.

When did beer enter into Europe?

When Julius Caesar conquered Egypt in 55 BC.

What is Nitrogen Catabolite Repression (NCR)? What three enzymes does it inhibit?

When the presence of NH3 inhibits a yeast cell's ability to use urea as a nitrogen source. It inhibits three pathways: Car1 (forms urea and ornithine from arginine), Dur3 (urea permease used for importing), and Dur1,2 (converts urea to free ammonia).

What is a key difference between how white wines and red wines are made?

White wines are made without the skins (where the anthocyans are present) whereas red wines are made both with the grapes and the skins.

What are Ice wines? How much juice do you get in comparison to normal grapes?

Wines that are produced from grapes that are purposefully left on the vine until they freeze such that sweet, concentrated syrup can be crushed from the shrivelled grapes. You only tend to get about 5-10% of the amount of juice in comparison to normal grapes.

Of the three types of organisms used for making bio-ethanol (Saccharomyces yeast, Zymomonas mobilis, Clostridium thermocellum), how do they compare with respect to their ethanol yield?

Yeast - High yield. They have an actual yield that is higher than their theoretical yield, given that as the ethanol concentration increases, the yeast will devote increasing amounts of ATP to survival processes as opposed to growth. Zymomonas - Actual yield of 97%. These bacteria utilize the Entner-Doudoroff pathway (as opposed to Embden-Meyerhof), producing only 1 ATP instead of 2. As such, they need to produce a lot of enzymes to keep up with the demand and will produce a lot of pyruvate (ready to be converted to ethanol) very quickly, devoting more carbon to ethanol producing. Clostridium - low ethanol yields.

Of the three types of organisms used for making bio-ethanol (Saccharomyces yeast, Zymomonas mobilis, Clostridium thermocellum), how they compare with respect to growth temperatures?

Yeast - They grow at 30°C Zymomonas - They need to be grown at a temperature of at least 37°C (to avoid fructan production), but can't really grow much higher than 42°C. Clostridium - These are thermophiles and thus, can ferment at high temperatures (which is great for saving on energy costs, given that you don't need to cool the tank as much during fermentation nor do you need to heat it as much when trying to distil the ethanol out (given that fermentation produces heat naturally).

Of the three types of organisms used for making bio-ethanol (Saccharomyces yeast, Zymomonas mobilis, Clostridium thermocellum), how do they compare with respect to the by-products they produce?

Yeast - They produce glycerol as their compatible solute (which you don't want) Zymomonas - They produce sorbitol as a compatible solute (not glycerol). They also have an enzyme called SacB (Levan Sucrase) which produces glucose and fructose from sucrose, but when working at a temperature below 37°C, it will produce fructan (a fructose polymer) as a by-product, which gums up the fermentation and will waste half of the carbon in the reaction. Clostridium - They make a lot of by-products

Of the three types of organisms used for making bio-ethanol (Saccharomyces yeast, Zymomonas mobilis, Clostridium thermocellum), how do they compare with respect to the carbon sources they can accept?

Yeast - can't take in any sugar polymers greater than trimers. They also can't metabolize pentoses. Zymomonas - can't take in any sugar polymers greater than trimers. They also can't metabolize pentoses. Clostridium - These can metabolize pentoses

Of the three types of organisms used for making bio-ethanol (Saccharomyces yeast, Zymomonas mobilis, Clostridium thermocellum), how do they compare with respect to ethanol tolerance?

Yeast - they have a high ethanol tolerance. They make Oleic acid (an 18 carbon fatty acid that is longer than normal membrane fatty acids), which increase their ethanol tolerance, but maintain fluidity via the cis-double bond. They also produce ergosterol, which helps with tolerance. Zymomonas - They have a high ethanol tolerance. They produce cis-vaccinic acid and hopanoids to aid in this (the synthesis of these molecules, unlike yeast, does not require oxygen). Clostridium - Low ethanol tolerance.

Why are late harvest wines tricky to ferment?

Yeast don't like the high sugar content of the Must, so there is generally a lot of residual sugar after the fermentation.

What are two ways of stopping malolactic fermentation?

You can carefully add potassium metabisulfite to kill off the lactic acid fermenters You can also add lysozyme to break the bonds that hold peptidoglycan bonds together.

How would you go about making a 25 Brix solution?

You would add 25 g sugar to 75 mL water

What is the difference between α and β glycosidic bonds?

α - The anomeric carbons are within the same plane β - The anomeric carbons are on opposite planes