Questions: Microbial Biotechnology and Industrial Applications

Question 1

A pharmaceutical company wants to produce a therapeutic antibody that requires glycosylation for proper folding and immune effector function. Which production host is most appropriate?

• A. Escherichia coli, because it grows fastest and produces the highest volumetric yields
• B. Mammalian cell lines such as CHO cells, because bacteria cannot perform the required glycosylation
• C. Any bacterial host — glycosylation can be added chemically after protein purification
• D. Yeast grown in minimal media, because yeast are cheaper than mammalian cells and grow rapidly

Answer: (B)

Bacteria lack the endoplasmic reticulum and Golgi apparatus required for eukaryotic-type N-glycosylation, a critical post-translational modification for many therapeutic antibodies. CHO (Chinese hamster ovary) cells perform human-compatible glycosylation and are the industry standard for therapeutic antibody production. Yeast can glycosylate proteins but produce high-mannose glycans that differ from human glycosylation patterns and can trigger immune responses. The choice of host organism is the first decision in bioprocess design precisely because not all hosts can produce all proteins in functional form.

Question 2

What is the primary purpose of fed-batch fermentation rather than providing all nutrients to a bioreactor at the start of cultivation?

• A. To reduce energy costs by slowing microbial growth and lowering oxygen demand
• B. To prevent metabolic overflow, where excess nutrients cause cells to produce toxic byproducts like acetate instead of the desired product
• C. To allow continuous sampling without disturbing cell density or pH
• D. To maintain sterility by reducing the frequency of nutrient additions from outside the bioreactor

Answer: (B)

When E. coli or yeast are given excess glucose, they grow fast but divert carbon into overflow metabolism — producing acetate (bacteria) or ethanol (yeast) as byproducts that acidify the medium, are toxic to cells, and divert carbon away from the target product. Fed-batch fermentation adds nutrients gradually, keeping cells below the overflow threshold while maintaining continuous growth and product synthesis. This prevents the productivity crash that occurs when byproduct accumulation becomes toxic, and dramatically improves yields of recombinant proteins.

Question 3

Bacteria like E. coli are generally unsuitable for producing therapeutic proteins that require glycosylation, even though they grow faster and are cheaper to maintain than mammalian cells.

• T. True
• F. False

Answer: True

E. coli grows roughly 50–100 times faster than mammalian cells and is far cheaper to feed and maintain. But fast and cheap are irrelevant if the product is non-functional. Bacteria do not have the secretory machinery to add N-linked or O-linked sugar chains to proteins in the patterns required for many human therapeutics. Insulin, which does not require glycosylation, can be produced in E. coli successfully. Erythropoietin, clotting factors, and monoclonal antibodies require glycosylation for activity or stability, and must be produced in eukaryotic hosts — at much higher cost and complexity.

Question 4

Bioremediation is primarily a theoretical application of microbial biotechnology with few proven real-world deployments at industrial or environmental scale.

• T. True
• F. False

Answer: False

Bioremediation is actively deployed at industrial and environmental scale. Pseudomonas and related bacteria are used to degrade petroleum hydrocarbons at oil spill sites; constructed wetland systems use microbial consortia to remove nitrogen and phosphorus from municipal wastewater; bioreactors with specialized microbial communities treat industrial effluents. The 2010 Deepwater Horizon spill, for example, involved large-scale application of hydrocarbon-degrading bacteria. While engineering challenges remain (especially for recalcitrant pollutants like chlorinated compounds), bioremediation is an established practice, not just a research prospect.

Question 5

What does metabolic engineering mean in the context of industrial microbiology, and why is it more effective than simply inserting the gene for a target product into a host organism?

Model answer: Metabolic engineering is the systematic redesign of a cell's metabolic network to optimize the production of a target compound. Simply inserting the gene for a target enzyme often yields low productivity because the cell distributes carbon through its natural metabolic pathways — many of which compete with or divert from the target pathway. Metabolic engineering involves: knocking out competing pathways that consume the same precursors, overexpressing rate-limiting enzymes in the target pathway, importing entirely new biosynthetic routes from other organisms, and balancing cofactor availability (NADPH, ATP) to sustain production. The result is a cell whose central metabolism is rewired to maximize flux through the target pathway, often yielding 10–100x higher product titers than simple gene expression.

A classic example: producing the antimalarial precursor artemisinic acid in yeast required not just inserting the biosynthetic genes from Artemisia annua but also engineering the yeast's sterol pathway, upregulating relevant reductases, and eliminating competing reactions — over 20 genetic modifications total. This level of systematic pathway optimization is what distinguishes metabolic engineering from basic recombinant protein expression.