Application of a mechanistic model as a tool for on-line monitoring of pilot scale filamentous fungal fermentation processes-The importance of evaporation effects.

A mechanistic model-based soft sensor is developed and validated for 550L filamentous fungus fermentations operated at Novozymes A/S. The soft sensor is comprised of a parameter estimation block based on a stoichiometric balance, coupled to a dynamic process model. The on-line parameter estimation block models the changing rates of formation of product, biomass, and water, and the rate of consumption of feed using standard, available on-line measurements. This parameter estimation block, is coupled to a mechanistic process model, which solves the current states of biomass, product, substrate, dissolved oxygen and mass, as well as other process parameters including kL a, viscosity and partial pressure of CO2 . State estimation at this scale requires a robust mass model including evaporation, which is a factor not often considered at smaller scales of operation. The model is developed using a historical data set of 11 batches from the fermentation pilot plant (550L) at Novozymes A/S. The model is then implemented on-line in 550L fermentation processes operated at Novozymes A/S in order to validate the state estimator model on 14 new batches utilizing a new strain. The product concentration in the validation batches was predicted with an average root mean sum of squared error (RMSSE) of 16.6%. In addition, calculation of the Janus coefficient for the validation batches shows a suitably calibrated model. The robustness of the model prediction is assessed with respect to the accuracy of the input data. Parameter estimation uncertainty is also carried out. The application of this on-line state estimator allows for on-line monitoring of pilot scale batches, including real-time estimates of multiple parameters which are not able to be monitored on-line. With successful application of a soft sensor at this scale, this allows for improved process monitoring, as well as opening up further possibilities for on-line control algorithms, utilizing these on-line model outputs. Biotechnol. Bioeng. 2017;114: 589-599. © 2016 Wiley Periodicals, Inc.

A novel model-based control strategy for aerobic filamentous fungal fed-batch fermentation processes.

A novel model-based control strategy has been developed for filamentous fungal fed-batch fermentation processes. The system of interest is a pilot scale (550 L) filamentous fungus process operating at Novozymes A/S. In such processes, it is desirable to maximize the total product achieved in a batch in a defined process time. In order to achieve this goal, it is important to maximize both the product concentration, and also the total final mass in the fed-batch system. To this end, we describe the development of a control strategy which aims to achieve maximum tank fill, while avoiding oxygen limited conditions. This requires a two stage approach: (i) calculation of the tank start fill; and (ii) on-line control in order to maximize fill subject to oxygen transfer limitations. First, a mechanistic model was applied off-line in order to determine the appropriate start fill for processes with four different sets of process operating conditions for the stirrer speed, headspace pressure, and aeration rate. The start fills were tested with eight pilot scale experiments using a reference process operation. An on-line control strategy was then developed, utilizing the mechanistic model which is recursively updated using on-line measurements. The model was applied in order to predict the current system states, including the biomass concentration, and to simulate the expected future trajectory of the system until a specified end time. In this way, the desired feed rate is updated along the progress of the batch taking into account the oxygen mass transfer conditions and the expected future trajectory of the mass. The final results show that the target fill was achieved to within 5% under the maximum fill when tested using eight pilot scale batches, and over filling was avoided. The results were reproducible, unlike the reference experiments which show over 10% variation in the final tank fill, and this also includes over filling. The variance of the final tank fill is reduced by over 74%, meaning that it is possible to target the final maximum fill reproducibly. The product concentration achieved at a given set of process conditions was unaffected by the control strategy. Biotechnol. Bioeng. 2017;114: 1459-1468. © 2017 Wiley Periodicals, Inc.

Locating the stem cell niche and tracing hepatocyte lineages in human liver.

UNLABELLED: We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser-capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase-deficient hepatocytes were clonal, suggesting an origin from a long-lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase-deficient hepatocytes were nonpathogenic. CONCLUSION: These data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins.

A methodological approach to tracing cell lineage in human epithelial tissues.

Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites.

An Industrial Perspective on Scale-Down Challenges Using Miniaturized Bioreactors.

Miniaturized stirred bioreactors (MSBRs) are gaining popularity as a cost-effective approach to scale-down experimentation. However, realizing conditions that reflect the large-scale process accurately can be challenging. This article highlights common challenges of using MSBRs for scale-down. The fundamental difference between oxygen mass transfer coefficient (kLa) and oxygen transfer rate scaling is addressed and the difficulty of achieving turbulent flow and industrially relevant tip speeds is described. More practical challenges of using MSBR systems for scale-down are also discussed, including the risk of vortex formation, changed volume dynamics, and wall growth. By highlighting these challenges, the article aims to create more awareness of these difficulties and to contribute to improved design of scale-down experiments.

A review of control strategies for manipulating the feed rate in fed-batch fermentation processes.

A majority of industrial fermentation processes are operated in fed-batch mode. In this case, the rate of feed addition to the system is a focus for optimising the process operation, as it directly impacts metabolic activity, as well as directly affecting the volume dynamics in the system. This review covers a range of strategies which have been employed to use the feed rate as a manipulated variable in a control strategy. The feed rate is chosen as the focus for this review, as it is seen that this variable may be used towards many different objectives depending on the process of interest, the characteristics of the strain, or the product being produced, which leads to different drivers for process optimisation. This review summarises the methods, as well as focusing on the different objectives for the controllers, and the choice of measured variables involved in the strategy. The discussion includes a summary of considerations for control strategy development.

Mechanistic Fermentation Models for Process Design, Monitoring, and Control.

Mechanistic models require a significant investment of time and resources, but their application to multiple stages of fermentation process development and operation can make this investment highly valuable. This Opinion article discusses how an established fermentation model may be adapted for application to different stages of fermentation process development: planning, process design, monitoring, and control. Although a longer development time is required for such modeling methods in comparison to purely data-based model techniques, the wide range of applications makes them a highly valuable tool for fermentation research and development. In addition, in a research environment, where collaboration is important, developing mechanistic models provides a platform for knowledge sharing and consolidation of existing process understanding.

Lymphoepithelial Cyst of the Pancreas.

Lymphoepithelial cyst (LEC) of the pancreas is an extremely rare, benign pancreatic cystic lesion that is difficult to differentiate preoperatively from other cystic pancreatic lesions. LEC may have malignant potential. Here, we describe a case of LEC of the pancreas - initially suspected to be a mucinous cyst neoplasm - in an elderly man presenting with abdominal pain, who went on to have a distal pancreatectomy and splenectomy. We also review the relevant literature and discuss implications for the diagnosis and management of this rare lesion.

Repeated negative biopsies in isolated high-grade cystic duct dysplasia with progression to adenocarcinoma.

Cystic and bile duct dysplasia is a rare histological finding, especially when found in the absence of an underlying malignancy. We report a patient who presented with jaundice and weight loss. Clinical and cytological evidence suggested a diagnosis of cholangiocarcinoma and the patient underwent a pancreatico-duodenectomy. Histopathological examination suggested a diagnosis of two foci of biliary dysplasia: cystic duct and lower common bile duct. Fifteen months later, the patient re-presented with signs of obstructive jaundice and biliary sepsis. Although CT scan revealed images highly indicative of metastatic disease, repeated biopsies failed to confirm this. Eventually a liver biopsy did reveal moderately differentiated adenocarcinoma, however oncological interventional was no longer appropriate and the patient was managed palliatively. This case report focuses on the current understanding of progression of biliary dysplasia.

Challenges in industrial fermentation technology research.

Industrial fermentation processes are increasingly popular, and are considered an important technological asset for reducing our dependence on chemicals and products produced from fossil fuels. However, despite their increasing popularity, fermentation processes have not yet reached the same maturity as traditional chemical processes, particularly when it comes to using engineering tools such as mathematical models and optimization techniques. This perspective starts with a brief overview of these engineering tools. However, the main focus is on a description of some of the most important engineering challenges: scaling up and scaling down fermentation processes, the influence of morphology on broth rheology and mass transfer, and establishing novel sensors to measure and control insightful process parameters. The greatest emphasis is on the challenges posed by filamentous fungi, because of their wide applications as cell factories and therefore their relevance in a White Biotechnology context. Computational fluid dynamics (CFD) is introduced as a promising tool that can be used to support the scaling up and scaling down of bioreactors, and for studying mixing and the potential occurrence of gradients in a tank.

Prevalence of hepatitis C-related cirrhosis in elderly Asian patients infected in childhood.

BACKGROUND & AIMS: Approximately 20% of hepatitis C virus (HCV) patients develop cirrhosis from infection after about 20 years. The proportion of patients developing cirrhosis for longer than 30 years after infection is unknown. Our objectives were to determine the prevalence of HCV-related cirrhosis in a population of Asian patients who were infected in childhood 20 to 80 years ago and compare this with the prevalence of cirrhosis in Caucasian patients referred to the same hospitals. METHODS: Retrospective analyses were performed of all patients who had detectable HCV-RNA levels and who attended local hospitals in northeast London between 1992 and 2003. Factors implicated in the development of cirrhosis were examined by multivariable analysis. RESULTS: A total of 143 adult Asian patients who had been infected with HCV for many decades were compared with 239 Caucasian patients. The prevalence of cirrhosis increased with age. Of Asian patients aged 61-80 years (n = 55) 78% had cirrhosis, whereas 25% of Caucasian patients aged 61-80 years (n = 55) had cirrhosis. Multivariable linear analysis revealed that fibrosis progression and age were similar in both groups and the difference in the prevalence of cirrhosis was not explained by any unique Asian characteristic other than prolonged infection. CONCLUSIONS: The prevalence of cirrhosis in patients with chronic HCV increases with increasing duration of infection. In Asian patients infected at birth, infection for over 60 years causes cirrhosis in 71% of infected individuals. Because relationship between the severity of fibrosis and age in Asian patients is similar to that seen in Caucasian patients it is likely that similar rates of cirrhosis will be seen in other patients who are infected for more than 60 years.