E: Questions

Question 1

Providing a single example, define a primary metabolite type 1?

Answer 1

Primary metabolites are growth-linked products (proportional growth rate and biomass) and are formed in the log phase. An example is alcohol.

Question 2

Providing a single example, define a secondary metabolite?

Answer 2

Secondary metabolites are non-growth-linked products, independent of mu, and are produced during the stationary phase. An example is antibiotics.

Question 3

What is the foodstuff known as Quorn?

Answer 3

A single cell protein (mycoprotein) product used as a human food meat substitute, made from the fungus, Fusarium graminearum, and grown in continuous culture

Question 4

What was the feedstuff known as Pruteen?

Answer 4

A single cell protein product made from the bacterium, Methylophilus (Pseudomonas) methylotrophus, ad grown in continuous culture for the animal feed market.

Question 5

What is the feedstuff known as BioProtein?

Answer 5

A single cell protein produced by growing Methylococcus capsulatus on methane. Due to impurities in the methane gas by longer alkanes (C2 and C3), other bacteria are added to the fermentor to remove these impurities: Alcaligenes acidovorans, Bacillus firmus and Bacillus brevis.

Question 6

Identify the key advantage of penicillin V over penicillin G. What type of penicillin is methicillin?

Answer 6

Penicillin V is orally available.
Methicillin is semi-synthetic.

Question 7

Does a bioassay measure the concentration or potency of an antibiotic?
Which on one is relevant to a clinician when calculating a dose of medicine for a patient? Explain why this is the case?

Answer 7

Potency
Potency: to permit accurate dosing (‘mg/kg’ calculation

Question 8

How would you detect the activity of an antibiotic during random screening procedures of soil microbes for metabolites of commercial interest?

Answer 8

Use agar diffusion assay format where an antagonistic reaction is set up between test antibiotic producer and a number of potentially susceptible pathogen test organisms (such as MRSA, S. aureus, P. aeruginosa, Candida etc.)

Question 9

Discriminate between a biosynthetic (natural) and a semi-synthetic penicillin, giving a single example in each case (explain the difference between). Provide one advantage offered by a semi-synthetic penicillin?

Answer 9

Biosynthetic entails directing the organisms to produce mainly a single type of penicillin by addition of a precursor substance to the organisms’ growth medium (e.g. penicillin G and phenylacetic acid). Semi-synthetic also involves a biosynthetic route, but followed by chemical or enzymatic substitution onto the 6-PA nucleus (e.g. methicillin). Advantages are oral availability and being able to combat resistant organisms.

Question 10

Explain the difference between a natural penicillin and a biosyntheitc penicillin?

Answer 10

If a fermentation is carried out without the addition to the growth medium of side-chain precursor, then the ‘natural penicillins’ are produced (for example, a proportion of peniciilins produced will be penicillin G, which has phenylacetic acid as a side chain, with the phenylacetic acid being derived from corn steep liquor). The purposeful addition of a side chain precursor to the growth medium will produce a ‘biosynthetic penicillin’, consisting largely of a single penicillin type.

Question 11

During random screening procedures of soil microbes for metabolites of commercial interest, how would you detect the activity of (alpha-) amylases, proteases and lipases? Briefly, explain the principle of the assays?

Answer 11

Amylases (starch agar; examine for clear zones around enzyme-producing colonies against a purple background after flooding plate with Lugol’s iodine. Enzymes produced by cells diffuse through the medium and depolymerise casein and starch), Proteases (casein agar; look for zones of clearing around colonies against a turbid background), Lipases (Tween 80/tributryn agar, look for zones of opacity)

Question 12

Briefly, describe the malting step in beer fermentation?

Answer 12

Conversion of starch into fermentable sugars (milling, soaking, spreading on malting floor). Hydrolytic enzymes attach starchy grain endosperm. Germinated seeds are then killed by kilning.

Question 13

The production of ales and lagers are different in a number of process aspects? List any two (more) of these differences?

Answer 13

S. cerevisiae var. cerevisiae for ales and S. cerevisiae var. carlsbergenesis (uvarum) for lagers. Ale yeasts are ‘top fermentors’ (12-18C) and lager yeasts are ‘bottom fermentors’ (8-12C – take longer). Maturation differences (ales take 1-3 days, while lagers take up to 3 weeks)

Question 14

Which yeasts are used in alcohol fermentation to produce (i) lagers and (ii) ales?

Answer 14

Lagers = Saccharomyces cerevisiae var carlsbergenesis (uvarum) and ales = S. cerevisiae var cerevisiae.

Question 15

Define a primary metabolite type 2?

Answer 15

partial-growth linked products (partly independent from the specific growth rate) - fermentation end-products of energy metabolism, including organic compounds produced as a result of an anaerobic fermentative process or resulting from an incomplete oxidative process

Question 16

What are the advantages and disadvantages of cell-based manufacture versus chemical manufacture?

Question 17

Explain what is meant by compressed yeast?

Answer 17

Compressed yeast (also called cake, wet, or fresh yeast) in which the water content of the yeast cream is further reduced by passing through a filter press or rotary vacuum filtration unit. Once pressed, the yeast cake is extruded through a rectangular nozzle that is cut into the proper length and weight.

Question 18

What is meant by ‘active dry yeast’?

Answer 18

Process begins as per process for compressed yeast (water content of the yeast cream is further reduced by passing through a filter press or rotary vacuum filtration unit), but the cake is extruded through perforated plates or screens in the form of thin spaghetti-like strands (then pelleted, dried and ground into small granules).

Question 19

Provide two advantages of modern penicillins compared with penicillin G?

Answer 19

Orally available, broad spectrum (more active against Gram negative organisms), more resistant to beta-lactamases.

Question 20

What is the principle of the penicillin bioassay?

Answer 20

A dish is seeded with a susceptible microorganism, Staphylococcus aureus. Holes are aseptically drilled into the medium and the wells are filled with test and standard solutions of penicillin. After incubation, zones of inhibited growth of the S. aureus are measured, a standard curve is constructed, and the value of the unknowns interpolated from this.

Question 21

Provide two reasons why the sweet wort is boiled during the mashing stage of beer fermentation?

Answer 21

To halt enzymatic processes; to pasteurise it; to precipitate ‘hot trub’; to dissolve any adjunct sugars.

Question 22

Is a SCP classified as a primary metabolite or a secondary metabolite? Explain?

Answer 22

SCPs are primarily primary metabolites [best representative of a product – in this case, the cell biomass itself – arising from the growth phase of a culture]. Secondary metabolites are non-growth-associated (mostly produced under nutrient-limited conditions in the stationary phase of growth).

Question 23

How does penicillin work?

Answer 23

Interferes with transpeptidase activity, blocking the assembly of bacterial peptidoglycan (that makes peptide crosslinks), and cells therefore become ‘leaky’

Question 24

Genus Bacillus, found in soil, is a well-recognised source of industrially useful amylase and protease enzymes. Given a sample of soil, briefly describe how you would specifically select for this genus?

Answer 24

Genus Bacillus are aerobic spore formers. Therefore, make an aqueous extract of the soil and heat for 10 minutes at 80C and incubate at 30C overnight on incubator media.

Question 25

There is a shortage of new antibiotics for human health coming through the drug development pipeline. Provide one more reason for this trend?

Answer 25

(i) technological (and discovery) challenges in developing new antibiotics; (ii) the highest value pharmaceutical markets are chronic diseases (such as cardiovascular and arthritis); antibiotics are used to treat acute (short term) disease (currently most individual antibiotics generate only about $200-$300 Mn pa); (iii) there are many generic antibiotics, with substitution possible by clinicians, and so the market is difficult from an economic perspective.

Question 26

How may one reduce the levels of RNA in a single cell protein food derived from fungi? Why is this step necessary?

Answer 26

Chemical treatment (base-catalysed hydrolysis by NH4OH, KOH or NaOH) or enzymatic (exogenous RNAase or endogenous RNAse/heat shock) methods.
High nucleic acid levels result in uric acid deposition in joints of humans, and gout