Lessons learned: use of WGS in real-time investigation of suspected intrahospital SARS-CoV-2 outbreaks.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been a continuing source of hospital-acquired infection and outbreaks. At Akershus University Hospital in Norway, traditional contact tracing has been combined with whole-genome sequencing (WGS) surveillance in real-time to investigate potential hospital outbreaks. AIM: To describe the advantages and challenges encountered when using WGS as a real-time tool in hospital outbreak investigation and surveillance during the SARS-CoV-2 pandemic. METHODS: Routine contact tracing in the hospital was performed for all healthcare workers (HCWs) who tested positive for SARS-CoV-2. Viral RNA from all positive patient and HCW samples was sequenced in real-time using nanopore sequencing and the ARTIC Network protocol. Suspected outbreaks involving five or more individuals with viral sequences were described. FINDINGS: Nine outbreaks were suspected based on contact tracing, and one outbreak was suspected based on WGS results. Five outbreaks were confirmed; of these, two outbreaks were supported but could not be confirmed by WGS with high confidence, one outbreak was found to consist of two different lineages, and two outbreaks were refuted. CONCLUSIONS: WGS is a valuable tool in hospital outbreak investigations when combined with traditional contact tracing. Inclusion of WGS data improved outbreak demarcation, identified unknown transmission chains, and highlighted weaknesses in existing infection control measures.

Investigation of intra-hospital SARS-CoV-2 transmission using nanopore whole-genome sequencing.

BACKGROUND: During the SARS-CoV-2 pandemic, healthcare workers (HCWs) are being exposed to infection both at work and in their communities. Determining where HCWs might have been infected is challenging based on epidemiological data alone. At Akershus University Hospital, Norway, several clusters of possible intra-hospital SARS-CoV-2 transmission were identified based on routine contact tracing. AIM: To determine whether clusters of suspected intra-hospital SARS-CoV-2 transmission could be resolved by combining whole genome sequencing (WGS) of SARS-CoV-2 with contact tracing data. METHODS: Epidemiological data were collected during routine contact tracing of polymerase chain reaction-confirmed SARS-CoV-2-positive HCWs. Possible outbreaks were identified as wards with two or more infected HCWs defined as close contacts who tested positive for SARS-CoV-2 less than three weeks apart. Viral RNA from naso-/oropharyngeal samples underwent nanopore sequencing in direct compliance to the ARTIC Network protocol. FINDINGS: Five outbreaks were suspected from contact tracing. Viral consensus sequences from 24 HCWs, two patients, and seven anonymous samples were analysed. Two outbreaks were confirmed, one refuted, and two remained undetermined. One new potential outbreak was discovered. CONCLUSION: Combined with epidemiological data, nanopore WGS was a useful tool for investigating intra-hospital SARS-CoV-2 transmission. WGS helped to resolve questions about possible outbreaks and to guide local infection prevention and control measures.

Staphylococcus aureus colonization during military service: a prospective cohort study.

OBJECTIVES: Staphylococcus aureus colonization leading to skin and soft-tissue infections (SSTI) are known challenges in crowded settings such as the military. The aim of the study was to establish and compare the prevalence of S. aureus colonization in recruits at enrolment and discharge after the first year of military service, and to investigate the prevalence of S. aureus SSTI. METHODS: All recruits entering first year of military service in January 2013 to be stationed at three garrisons in the northern part of Norway were invited to join this prospective cohort study. Swabs were taken from nose, throat and perineum. Staphylococcus aureus was identified using standard culturing methods. Methicillin resistance was determined by a cefoxitin disc diffusion test. RESULTS: Of the 923 eligible recruits, 512 were included at enrolment; 265/512 (52%) were also screened at discharge. Staphylococcus aureus colonization was high, and increased significantly during military service (166/265 versus 224/265, p < 0.001) mainly caused by increase in throat colonization alone or in combination with nasal colonization. All S. aureus isolates were susceptible to methicillin. SSTI was self-reported in 7/265 (3%) recruits, of which only one was confirmed by a military physician. CONCLUSION: Staphylococcus aureus colonization increased during military service, but there were few confirmed reports of SSTI. Inclusion of throat swab provides important information as ∼20% of the recruits were only positive in their throat. Further analyses need to be performed to investigate if the increase in colonization is caused by specific S. aureus stains.

Emerging multidrug-resistant Bengal Bay clone ST772-MRSA-V in Norway: molecular epidemiology 2004-2014.

A multidrug-resistant, methicillin-resistant Staphylococcus aureus (MRSA) clone, PVL-positive ST772-MRSA-V, named the Bengal Bay clone, is emerging worldwide. In Norway, where MRSA prevalence is low, a sudden increase in ST772-MRSA-V initiated a nationwide molecular epidemiological study. Clinical data were obtained from the Norwegian Surveillance System for Communicable Diseases (MSIS). S. aureus isolates were characterised by antibiotic susceptibility profiles and comprehensive genotyping (spa typing, MLVA, DNA microarray). ST772-MRSA was detected in 145 individuals during 2004-2014, with 60% of cases occurring in 2013-2014. Median age was 31 years and male/female ratio 1.16. The majority had a family background from the Indian subcontinent (70%). MRSA acquisition was mainly reported as unknown (39%) or abroad (42%), the latter associated with a home-country visit (59%), tourism (16%), and immigration (13%). Clinical infection was present in 75%, predominantly by SSTI (83%), 18% were admitted to hospital and 42% were linked to small-scale outbreaks (n = 25). All isolates were multidrug-resistant. Most isolates were resistant to erythromycin, gentamicin and norfloxacin. Genotyping revealed a conserved clone predominated by spa type t657 (83%), MLVA-type 432 (67%) and the genes lukF/S, sea, sec/sel, egc, scn, cna, ccrAA/ccrC, agrII and cap5. A few untypical ccr gene combinations were detected. Bengal Bay isolates have likely been imported on several occasions and revision of infection control guidelines may prevent further spread.

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

BACKGROUND: The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. AIM: To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. METHODS: A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. FINDINGS: All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. CONCLUSION: MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates.

Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/blaCMY-2 resistance plasmids.

OBJECTIVES: In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). METHODS: Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a blaCMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and blaCMY-2. All IncK/blaCMY-2-positive isolates were analysed with WGS-based bioinformatics tools. RESULTS: Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/blaCMY-2 plasmid variants highly similar to the IncK/blaCMY-2 plasmid present in the poultry isolates. CONCLUSIONS: Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota.

Heat-resistant, extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in endoscope-mediated outbreak.

BACKGROUND: We describe an outbreak with an extended-spectrum ß-lactamase-producing Klebsiella pneumoniae strain in an intensive care unit in a secondary care hospital in Norway. The outbreak source was a fibreoptic intubation endoscope in which the outbreak strain survived despite chemothermal disinfection in a decontaminator designated for such use. The genetic marker clpK, which increases microbial heat resistance, has previously been described in K. pneumoniae outbreak strains. AIM: To investigate the role of clpK in biofilm formation and heat-shock stability in the outbreak strain. METHODS: The outbreak investigation was done by review of clinical records, screening of patients and culture from intubation endoscopes and bronchoscopes. Amplified fragment length polymorphism was used to identify the outbreak strain. clpK detection was performed by polymerase chain reaction, followed by mutant construction and heat-shock assays. FINDINGS: Five patients and one intubation endoscope contained K. pneumoniae with the same amplified fragment length polymorphism pattern. The outbreak strain contained the clpK genetic marker, which rendered the strain its increased heat resistance. The survival rate of the strain grown as biofilm following heat treatment was also strongly dependent on clpK. CONCLUSION: Although clpK has been associated with clinical isolates of K. pneumoniae in earlier outbreaks, this is the first time that a ClpK-producing strain has been isolated from an environmental outbreak source. Heat resistance of certain K. pneumoniae strains may facilitate survival in biofilms on medical equipment and hence increase the potential of those strains to persist and disperse in the hospital environment.

Reduced Socs3 expression in adipose tissue protects female mice against obesity-induced insulin resistance.

AIMS/HYPOTHESIS: Inflammation in obesity increases the levels of the suppressor of cytokine signalling-3 (SOCS3) protein in adipose tissue, but the physiological importance of this protein in regulating whole-body insulin sensitivity in obesity is not known. METHODS: We generated Socs3 floxed (wild-type, WT) and Socs3 aP2 (also known as Fabp4)-Cre null (Socs3 AKO) mice. Mice were maintained on either a regular chow or a high-fat diet (HFD) for 16 weeks during which time body mass, adiposity, glucose homeostasis and insulin sensitivity were assessed. RESULTS: The HFD increased SOCS3 levels in adipose tissue of WT but not Socs3 AKO mice. WT and Socs3 AKO mice had similar body mass and adiposity, assessed using computed tomography (CT) imaging, irrespective of diet or sex. On a control chow diet there were no differences in insulin sensitivity or glucose tolerance. When fed a HFD, female but not male Socs3 AKO mice had improved glucose tolerance as well as lower fasting glucose and insulin levels compared with WT littermates. Hyperinsulinaemic-euglycaemic clamps and positron emission tomography (PET) imaging demonstrated that improved insulin sensitivity was due to elevated adipose tissue glucose uptake. Increased insulin-stimulated glucose uptake in adipose tissue was associated with enhanced levels and activating phosphorylation of insulin receptor substrate-1 (IRS1). CONCLUSIONS/INTERPRETATION: These data demonstrate that inhibiting SOCS3 production in adipose tissue of female mice is effective for improving whole-body insulin sensitivity in obesity.

Transgenic models--a scientific tool to understand exercise-induced metabolism: the regulatory role of AMPK (5'-AMP-activated protein kinase) in glucose transport and glycogen synthase activity in skeletal muscle.

The AMPK (5'AMP-activated protein kinase) is becoming recognized as a critical regulator of energy metabolism. However, many of these effects in muscle metabolism have been ascribed to AMPK based on the use of the unspecific activator AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside). Using mouse models in which AMPK activity has been specifically blocked (kinase dead) or knocked out we and others have been able to conduct studies gaining more conclusive data on the role of AMPK in muscle metabolism. In this mini-review focus is on AMPK and its regulatory role for glucose transport and GS (glycogen synthase) activity in skeletal muscle, indicating that AMPK is a GS kinase in vivo which might influence GS activity during exercise and that AMPK is involved in AICAR/hypoxia-induced glucose transport but not or only partially in contraction-stimulated glucose transport.

Signalling to glucose transport in skeletal muscle during exercise.

Exercise-induced glucose uptake in skeletal muscle is mediated by an insulin-independent mechanism. Although the signalling events that increase glucose transport in response to muscle contraction are not fully elucidated, the aim of the present review is to briefly present the current understanding of the molecular signalling mechanisms involved. Glucose uptake may be regulated by Ca++-sensitive contraction-related mechanisms possibly involving protein kinase C, and by mechanisms that reflect the metabolic status of the muscle and may involve the AMP-activated protein kinase. Furthermore the p38 mitogen activated protein kinase may be involved. Still, the picture is incomplete and a substantial part of the exercise/contraction-induced signalling mechanism to glucose transport remains unknown.

Insulin signalling: effects of prior exercise.

After the discovery and clinical use of insulin for treatment of diabetes it became clear that some of the biological effect of insulin was dependent on the circumstances under which it was given. Relevant for this review is the notion that physical activity, in addition to its own direct metabolic effects also markedly affects the ability of insulin to stimulate a range of metabolic processes. More specifically, during and for a prolonged period after, exercise elicits effects on processes such as insulin-induced muscle glucose uptake and glucose metabolism which influence systemic glucose homeostasis. These phenomena are probably responsible for the improvement in glucose homeostasis and metabolic control that typically occurs with exercise in people with insulin resistance and probably contributes to the reduced risk for development of type 2 diabetes in individuals who engage in regular exercise. Here we focus on the influence of a single bout of exercise on the action of insulin on processes such as glucose uptake and glucose storage in skeletal muscle.

A possible role for AMP-activated protein kinase in exercise-induced glucose utilization: insights from humans and transgenic animals.

Exercise-induced glucose uptake in skeletal muscle is mediated by an insulin-independent mechanism, but the actual signals to glucose transport in response to muscle contraction have not been identified. The 5'-AMP-activated protein kinase (AMPK) has emerged as a putative mediator of contraction-induced glucose transport, although no conclusive evidence has been provided so far. Recent experiments in AMPK transgenic mice suggest that glucose transport induced by 5-amino-4-imidazolecarboxamide riboside (AICAR) or hypoxia is mediated by AMPK. In contrast, contraction-induced glucose transport in rodent skeletal muscle induced by electrical stimulation in vitro or in situ is not influenced or is only partially reduced by abolishing both or one of the catalytic AMPK subunits. This is compatible with exercise studies done in humans, where no tight correlation is found between AMPK activity and glucose uptake during exercise. Taken together, these results question an essential role of AMPK in exercise-induced glucose uptake and imply that one or more additional pathways are involved in mediating glucose transport in skeletal muscle during exercise.

Physiological role of AMP-activated protein kinase (AMPK): insights from knockout mouse models.

AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To understand better the physiological role of the catalytic AMPK subunit isoforms, we generated two knockout mouse models with the alpha1 (AMPK alpha 1(-/-)) and alpha 2 (AMPK alpha 2(-/-)) catalytic subunit genes deleted. No defect in glucose homoeostasis was observed in AMPK alpha 1(-/-) mice. On the other hand, AMPK alpha 2(-/-) mice presented high plasma glucose levels and low plasma insulin concentrations in the fed period and during the glucose tolerance test. Nevertheless, in isolated AMPK alpha 2(-/-) pancreatic islets, glucose-stimulated insulin secretion was not affected. Surprisingly, AMPK alpha 2(-/-) mice were insulin-resistant and had reduced muscle glycogen synthesis as assessed in vivo by the hyperinsulinaemic euglycaemic clamp procedure. Reduction of insulin sensitivity and glycogen synthesis were not dependent on the lack of AMPK in skeletal muscle, since mice expressing a dominant inhibitory mutant of AMPK in skeletal muscle were not affected and since insulin-stimulated glucose transport in incubated muscles in vitro was normal in AMPK alpha 2(-/-) muscles. Furthermore, AMPK alpha 2(-/-) mice have a higher sympathetic tone, as shown by increased catecholamine urinary excretion. Increased adrenergic tone could explain both decreased insulin secretion and insulin resistance observed in vivo in AMPK alpha 2(-/-) mice. We suggest that the alpha2 catalytic subunit of AMPK plays a major role as a fuel sensor by modulating the activity of the autonomous nervous system in vivo.

Evaluation of the potential effects of equalization on the performance of biological phosphorus removal systems.

Experimental data confirming that the phosphorus removal efficiency in biological excess phosphorus removal (BEPR) systems temporarily decreases when the amount of volatile fatty acids (VFAs) added in the anaerobic phase is suddenly increased are presented. This decrease in efficiency results from the fact that acetate uptake is a rapid process and that the phosphate concentration at the end of the anaerobic phase increases rapidly. Because of the nonlinear dependence of the phosphate uptake rate on the poly-beta-hydroxyalkanoate (PHA) content of phosphate-accumulating organisms (PAOs), the increase in PAO PHA content associated with VFA uptake is not able to cause a proportional increase in the rate of phosphate uptake. This causes a temporary imbalance between phosphate release and uptake, leading to lower phosphate removal efficiency. The VFA loading to full-scale BEPR systems is not constant throughout the day, and temporary imbalances such as the ones imposed in the batch tests can occur in full-scale systems. The effect of diurnal variations in loading was demonstrated through simulation of the behavior of an A/OTM system receiving a time-variable influent. Equalization is proposed as a method to diminish the potential for imbalances between phosphate release and uptake by avoiding sudden increases of VFA loading to the plant. Significant improvements in the effluent quality from the simulated system were achieved using equalization. The improvements were greater when the influent contained VFAs than when the VFAs were formed by fermentation in the anaerobic zone. The simulations suggested that it may be possible to decrease the amount of phosphorus discharged by a factor as high as 4 through use of concentration equalization. When both flow and concentration equalization were used, the total amount of phosphorus discharged was decreased by a factor of 8. Equalization can be used, in concert with other strategies for preservation of the PHA content of PAOs under periods of low loadings, to minimize the magnitude of Monday phosphate peaks.

State estimation for a biological phosphorus removal process using an asymptotic observer.

This study investigated the use of an asymptotic observer for state estimation in a continuous biological phosphorus removal process. The estimated states are the concentration of heterotrophic, autotrophic and phosphorus accumulating organisms, polyphosphate, glycogen and PHA. The reaction scheme describing the process was simplified from a combined ASM 1-Delft model. Three examples were investigated: operation at steady state, operation at steady state with a random white-noise in the measurements and operation with a ramp disturbance. In each case, the estimation was quite accurate even if the convergence, driven by the dilution rate, was slow (from 15 to 60 days). The propagation of the measurement noise and a bias in the estimation of glycogen and PHA could be the result of the high condition number of one of the matrices used in the algorithm of the asymptotic observer for the aerated tanks.

Estimation of kinetic parameters in a structured yeast model using regularisation.

In this work, a procedure for estimating kinetic parameters in biochemically structured models was developed. The approach is applicable when the structure of a kinetic model has been set up and the kinetic parameters should be estimated. The procedure consists of five steps. First, initial values were found in or calculated from literature. Hereafter using sensitivity analysis the most sensitive parameters were identified. In the third step physiological knowledge was combined with the parameter sensitivities to manually tune the most sensitive parameters. In step four, a global optimisation routine was applied for simultaneous estimation of the most sensitive parameters identified during the sensitivity analysis. Regularisation was included in the simultaneous estimation to reduce the effect of insensitive parameters. Finally, confidence intervals for the estimated parameters were calculated. This parameter estimation approach was demonstrated on a biochemically structured yeast model containing 11 reactions and 37 kinetic constants as a case study.

A biochemically structured model for Saccharomyces cerevisiae.

A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S(f), as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.

Activation control of pur gene expression in Lactococcus lactis: proposal for a consensus activator binding sequence based on deletion analysis and site-directed mutagenesis of purC and purD promoter regions.

A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between -79 and -70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined -10 regions but lack sequences resembling -35 regions for sigma70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the purC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea that purC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

Regulation of a continuous yeast bioreactor near the critical dilution rate using a productostat.

Regulation of a continuous bioreactor with Saccharomyces cerevisiae is investigated. A number of different sensors are evaluated for this purpose and the process dynamics is investigated around the critical dilution rate. A sensor for reducing gas concentration in exhaust gases is selected for regulating the substrate flow rate. Closed loop identification experiments are carried out to enable identification of the process dynamics near the critical diluton rate. Due to the time-varying nature of this process an adaptive regulator seems to be a promising tool for providing good regulatory and setpoint tracking performance. A simple third order model is used for a model based control design with a Linear Quadratic (LQ)-regulator. The LQ-regulator performs well experimentally, both in an adaptive version where the model parameters are updated on-line, and in a non-adaptive version. During the test the process is exposed to a large disturbance in substrate feed concentration and to a small setpoint disturbance. The proposed regulator is a practical realisation of a productostat where the product in this case is an undesired primary metabolite. Thus, this paper demonstrates a more general principle of utilizing metabolic overflow metabolism for directing fluxes through a desired metabolic pathway. This principle is applicable in the presented form, if a (by-)product can be measured on-line.

Continuous cultivation start-up control--an experimental investigation.

A control strategy to avoid development of synchronous growth in carbohydrate limited Saccharomyces cerevisiae cultivations is proposed and experimentally investigated. The basic idea is to control the metabolic flux through the pathways by manipulating the substrate feed rate to keep the ethanol concentration at a low level. An adaptive and a fixed parameter controller were investigated experimentally. Both controllers were initialized at the target conditions for the continuous cultivation, where the uncontrolled process is known to be marginally stable. The latter fact renders it unfeasible to attempt open loop operation at the critical dilution rate. The adaptive controller turned out to be superior to the fixed parameter controller. The superiority of the adaptive controller is ascribed to its ability to identify the process under varying cell activity. The obtained experimental results demonstrate that the desired operating point is reproducibly obtainable. However, after prolonged operation under different types of disturbances the yeast seemed to adapt towards an increased respiratory activity for the same low level of ethanol in the medium.