Question 1

Crabtree effect phenotype: in the presence of oxygen, how does a Crabtree-positive microorganism (e.g., Saccharomyces cerevisiae) behave at high sugar concentrations?

Accepted Answer

Introduction / Context: The Crabtree effect describes aerobic fermentation: certain yeasts preferentially ferment glucose to ethanol at high sugar concentrations despite sufficient oxygen for respiration. This behavior influences reactor design, oxygen transfer, and product spectrum in industrial fermentations like brewing and bioethanol production. Given Data / Assumptions: Organism exhibits Crabtree positivity (e.g., S. cerevisiae). Oxygen is available but not necessarily limiting. Glucose concentration is high. Concept / Approach: At high glucose, glycolytic flux saturates downstream respiratory pathways, and regulatory networks (e.g., glucose repression) down-regulate respiration. Carbon is diverted to ethanol formation with ATP yield per glucose lower than respiration but higher volumetric rates due to glycolysis throughput. As glucose drops, cells shift back to respiratory metabolism (diauxic shift if ethanol is later consumed). Step-by-Step Solution: High glucose triggers glucose repression of respiratory genes and activates fermentation pathways.Even with oxygen present, pyruvate predominantly converts to ethanol (and CO2) rather than entering the TCA cycle extensively.Observed outcomes: high ethanol production, heat release, and oxygen uptake below purely respiratory demand. Verification / Alternative check: Batch profiles show ethanol accumulation during early high-glucose phase under aerobic conditions, followed by ethanol consumption after glucose depletion (diauxic growth), confirming the effect. Why Other Options Are Wrong: Low sugar scenarios do not typically elicit the Crabtree fermentation response.Pure respiration at high sugar conflicts with glucose repression dynamics. Common Pitfalls: Confusing Crabtree (aerobic fermentation due to high glucose) with Pasteur effect (oxygen inhibiting fermentation); assuming oxygen presence guarantees respiration dominance. Final Answer: Ferments at high sugar even in the presence of dissolved oxygen

Question 2

Organic acid production patterns: in many batch fermentations, acid formation is growth-associated initially and then continues during a non-growth phase. What is a plausible mechanistic reason for this behavior?

Accepted Answer

Introduction / Context: Product formation kinetics are often classified as growth-associated, non-growth-associated, or mixed (Luedeking–Piret model). Many organic acids (e.g., lactic, acetic) show significant formation during growth and can continue accumulating as growth slows due to stress and energy-spilling phenomena. Given Data / Assumptions: Batch culture with rising acid concentration and falling pH. Growth rate declines due to product inhibition and nutrient shifts. Acid formation persists even after biomass increase slows. Concept / Approach: Organic acids can partially uncouple energy generation (catabolism) from biomass synthesis (anabolism). Cells continue metabolizing substrate, maintaining redox balance and ATP turnover by excreting acid end-products when biosynthesis is inhibited. In Luedeking–Piret terms, the product rate r_p = α μ X + β X includes a non-growth-associated term (β X) that remains significant as μ decreases. Step-by-Step Solution: Early phase: high μ drives α μ X (growth-associated formation).As acid accumulates, pH drops; growth becomes stress-limited.Cells channel carbon to acid to maintain redox and energy balance (β X term persists).Therefore, production continues in a non-growth-associated manner. Verification / Alternative check: Plotting product vs. biomass often shows a nonzero intercept and slope, indicating mixed kinetics. Controlled pH runs reduce the non-growth component, supporting the uncoupling explanation. Why Other Options Are Wrong: Rising biomass yield over time is atypical under increasing stress.Classifying all organic acids as secondary metabolites is incorrect; many are primary products of central metabolism.High acid levels generally inhibit growth rather than stimulate it. Common Pitfalls: Equating non-growth-associated formation with stationary-phase only; overlooking the role of pH control and redox balancing in acid pathways. Final Answer: Organic acids uncouple catabolism from anabolism, sustaining formation even as growth slows

Question 3

Activated sludge process: why is biomass recycled from the secondary clarifier back to the aeration basin?

Accepted Answer

Introduction / Context: The activated sludge process for wastewater treatment relies on a dense, active microbial community to remove organics and nutrients. Recycling settled biomass (return activated sludge, RAS) from the clarifier to the aeration basin is fundamental to maintaining treatment performance. Given Data / Assumptions: Aeration basin followed by secondary clarification. Settled sludge is partially returned (RAS) and partially wasted (WAS). Objective: sustain a target mixed liquor suspended solids (MLSS) and sludge age. Concept / Approach: Returning biomass keeps the aeration basin rich in active organisms, which boosts reaction rates and removal efficiency. By controlling RAS and WAS, operators manage sludge retention time (SRT), which influences nitrification, settling properties, and overall stability. Efficient recycling also reduces the volume of sludge requiring disposal per unit of treatment by optimizing biomass growth and decay dynamics. Step-by-Step Solution: Recycle increases MLSS → higher volumetric removal rates.SRT control via RAS/WAS balances growth vs. decay, improving process stability.Optimized solids handling reduces net sludge production for disposal. Verification / Alternative check: Plant data show deterioration in effluent quality when RAS is interrupted, and restoration upon resuming recycle; modeling confirms sensitivity of effluent BOD/TSS to MLSS and SRT settings. Why Other Options Are Wrong: Each single-option rationale is true but incomplete; collectively they capture the full purpose, hence “All of the above.” Common Pitfalls: Confusing RAS (process control) with WAS (inventory control/disposal); neglecting impact of RAS flow on clarifier hydraulic loading and sludge blanket stability. Final Answer: All of the above

Question 4

Compared with batch culture, what is a key combined advantage of continuous culture for manufacturing integration and cell physiology control?

Accepted Answer

Introduction / Context: Continuous culture maintains steady-state conditions for extended periods, offering operational and biological advantages that can improve throughput and consistency in suitable applications. Given Data / Assumptions: Chemostat or perfusion systems with robust contamination control. Upstream and downstream unit operations can be aligned to continuous flow. Organism can sustain steady state without genetic drift or washout. Concept / Approach: Because nutrient and waste conditions are constant, cell physiology stabilizes, enabling reproducible product quality. Operationally, avoiding frequent turnarounds reduces downtime and cleaning cycles. Moreover, continuous feed and harvest can be synchronized with continuous clarification and chromatography strategies to streamline manufacturing. Step-by-Step Solution: Set dilution rate to the desired specific growth rate to hold cells at a defined physiological state.Sustain steady inputs/outputs to integrate with continuous downstream processing.Benefit from reduced start-stop transitions that consume labor and time in batch. Verification / Alternative check: Case studies show improved volumetric productivity and lot-to-lot consistency when continuous operation is feasible; however, implementation depends on contamination control and regulatory strategy. Why Other Options Are Wrong: Each individual statement is correct but partial; the best answer aggregates these advantages. Common Pitfalls: Underestimating sterility and genetic stability challenges; assuming continuous is universally superior without process-specific evaluation. Final Answer: All of the above

Question 5

Why are industrial baker's yeast (Saccharomyces cerevisiae) cultivations typically run in fed-batch reactors rather than simple batch? Explain the role of glucose level in shifting yeast between respiration and ethanol-forming fermentation.

Accepted Answer

Introduction / Context: Fed-batch is the workhorse mode for producing baker’s yeast and many yeast-derived products. The core reason relates to how extracellular glucose concentration controls the metabolic state of Crabtree-positive yeasts such as Saccharomyces cerevisiae, toggling between respiration (biomass-oriented) and ethanol-forming fermentation that can inhibit growth at high sugar levels. Given Data / Assumptions: Saccharomyces cerevisiae shows the Crabtree effect: at high glucose it ferments even with oxygen present. Ethanol accumulation reduces specific growth rate and yield on substrate. Glucose setpoint can be controlled by feeding in fed-batch (low but non-zero). Concept / Approach: Keeping extracellular glucose low biases metabolism toward respiratory growth with high biomass yields and minimal ethanol. Batch charges often overshoot glucose, provoking aerobic fermentation and ethanol accumulation. Fed-batch solves this by continuously or intermittently adding concentrated feed while oxygen is supplied to meet demand. Step-by-Step Solution: 1) If initial glucose is high (simple batch), S. cerevisiae triggers overflow metabolism → ethanol.2) Ethanol competes as a carbon sink and inhibits cells, depressing biomass yield.3) In fed-batch, glucose is dosed to a low setpoint (e.g., near zero), forcing respiration and maximizing biomass formation.4) Outcome: higher cell density, better oxygen utilization, and less inhibitory by-product. Verification / Alternative check: Oxygen uptake rate and CO2 evolution profiles in glucose-limited fed-batch confirm respiratory metabolism (low ethanol), whereas batch with excess glucose shows ethanol spikes and diauxic shifts upon ethanol re-consumption. Why Other Options Are Wrong: Option A and B invert the condition; fermentation and ethanol formation are associated with high, not low, glucose in Crabtree-positive yeast. Option D cannot be correct because A and B are false. Option E is wrong because C states the established rationale. Common Pitfalls: Confusing Pasteur and Crabtree effects; also assuming any feed rate works—overfeeding in fed-batch recreates batch-like glucose excess. Final Answer: Yeast cells respire at low glucose concentrations, so feed keeps glucose low to avoid ethanol inhibition

Question 6

Why are monoclonal antibodies (mAbs) from hybridoma or CHO cultures typically produced in fed-batch mode? Focus on how glucose feeding limits lactate accumulation and supports productivity.

Accepted Answer

Introduction / Context: Most industrial monoclonal antibody (mAb) processes use fed-batch operation with mammalian cells (hybridoma or CHO). A key reason is metabolic: excessive glucose drives aerobic glycolysis with high lactate formation, which can depress pH control, osmolarity, and specific productivity. Controlled feeding maintains glucose in a narrow band to limit lactate build-up and sustain viable, productive cultures. Given Data / Assumptions: Mammalian cells exhibit a Warburg-like effect: at high glucose, they produce lactate even under aerobic conditions. High lactate and acidification reduce viability and mAb quality attributes if unmanaged. Fed-batch allows tight control of extracellular glucose concentration. Concept / Approach: By delivering nutrient feeds gradually, fed-batch prevents large glucose spikes. The culture consumes glucose close to its uptake rate, reducing the driving force for overflow metabolism to lactate. This stabilizes pH control (less base addition), improves viable cell days, and supports sustained antibody secretion phases. Step-by-Step Solution: 1) Identify the metabolic issue: high glucose → high lactate via aerobic glycolysis.2) Choose a mode that avoids spikes: fed-batch with feedback or predefined boluses.3) Observe improved metrics: lower lactate, higher viability, longer production window, better titer.4) Conclude that feed strategy is central to modern mAb platforms. Verification / Alternative check: Process data commonly show lactate peaking early in batch but remaining controlled in fed-batch; some platforms harness lactate consumption phases when glucose is held low later in culture. Why Other Options Are Wrong: Option A misidentifies the sugar and mechanism; maltose is not the typical driver. Option C is the inverse of reality; high glucose favors lactate, not pure respiration. Option D is incorrect because B is valid; Option E ignores the dominant metabolic motivation. Common Pitfalls: Assuming more glucose always raises productivity; beyond a point, it worsens lactate and osmolality. Final Answer: Hybridoma cells yield higher lactate at high glucose concentrations, so controlled feeding reduces lactate

Fermentation Reactors Questions