Wastewater as a resource: a unique approach to achieving energy sustainability.

A wastewater-treatment flowsheet was developed to integrate uniquely designed biological processes with physical-chemical unit processes, allowing conversion of the organic carbon in the wastewater to methane, the removal and recovery of phosphorus and nitrogen from the wastewater, and the production of water suitable for reuse. In the flowsheet, energy is derived from the wastewater by first shunting a large fraction of the organic carbon in the wastewater to a solids slurry which is treated via anaerobic digestion. The anaerobic digestion system consists of focused pulsed (FP) pretreatment coupled to anaerobic membrane bioreactors (MBRs). Computer modelling and simulation results are used to optimize design of the system. Energy generation from the system is maximized and costs are reduced by using modest levels of recycle flow from the anaerobic MBRS to the FP pretreatment step.

Treating municipal wastewater with the goal of resource recovery.

A new municipal wastewater treatment flowsheet was developed with the objectives of energy sustainability, and water and nutrient recovery. Energy is derived by shunting a large fraction of the organic carbon in the wastewater to an anaerobic digestion system. Aerobic and anaerobic membrane bioreactors play a key role in energy recovery. Phosphorus and nitrogen are removed from the wastewater and recovered through physical-chemical processes. Computer modeling and simulation results together with energy balance calculations, imply the new flowsheet will result in a dramatic reduction in energy usage at lower treatment plant capital costs in comparison to conventional methods.

Use of a simple mathematical model to predict the behavior of Escherichia coli overproducing beta-lactamase within continuous single- and two-stage reactor systems.

A simple mathematical model is developed to help explain the complex population dynamics of an Escherichia coli host-plasmid expression/excretion system for beta-lactamase within single- and two-stage reactors. The model successfully integrates the individual regulatory (tac promoter induction), genetic (runaway plasmid replication), and population dynamics (culture instability) aspects of the system. The model predicts, and experiment confirms, that high-level beta-lactamase production and excretion cannot be easily maintained in single-stage reactors using the current plasmid construction. Stable target protein production and excretion is mathematically predicted, and experimentally confirmed, within two-stage reactors. The model is used to provide insight into engineering a more stable host-vector expression/excretion system for use in single-stage reactors.

Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of beta-lactamase from Escherichia coli.

Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.

Escherichia coli host cell modifications in continuous culture affecting heterologous protein overproduction: a population dynamics study.

There are many published studies of plasmid segregational instability in Escherichia coli in the literature. However, the formation of plasmid-free segregants can be controlled by the addition of selective chemical agents like antibiotics. This solution has become commonplace in both the laboratory and industry. On the other hand, host cell modifications, which result in low production of plasmid-encoded protein and lead to loss of culture productivity, have not been adequately addressed. Continuous culture of an inducible (ptac) Escherichia coli vector containing strain, RB791(pKN), was characterized by strong dynamic changes in the cell population and product (beta-lactamase) expression. Long-term cultivation resulted in the loss of high-level production of beta-lactamase. Loss of productivity was not due to the formation of plasmid-free cells or structural modifications to the plasmid; instead, continuous operation resulted in a culture dominated by irreversibly altered, low-producing cells. Two distinct classes of lac- mutants which inhibited induction were identified (Y- and I(s)).