<p>Zemlji&scaron;te predstavlja bogat izvor različitih mikroorganizama čiji<br />produkti metabolizma mogu biti od izuzetnog značaja za čoveka.<br />Dosada&scaron;nja ispitivanja mikrobnog diverziteta u zemlji&scaron;tu su<br />otkrila bogati biosintetski potencijal za proizvodnju novih<br />prirodnih proizvoda kod velikog broja mikroorganizama, naročito<br />kada je u pitanju klasa Actinobacteria. Među zemlji&scaron;nim izolatima,<br />rod Streptomyces prednjači po broju identifikovanih bioaktivnih<br />molekula u odnosu na sve ostale bakterije. Stoga je jedan od<br />ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz<br />rizosfera medicinski značajnih biljaka sakupljenih na teritoriji<br />Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica<br />dioica) i ispitivanje njihovog antifungalnog potencijala na različite<br />vrste kandida. Morfolo&scaron;ki različiti izolati (ukupno 103) su izolovani<br />iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve<br />različite podloge i dve procedure za ekstrakciju su kori&scaron;ćene da bi<br />se pospe&scaron;ila detekcija antifungalnih jedinjenja. Ispitan je uticaj<br />ukupno 412 ekstrakata na rast Candida albicans disk difuzionim<br />esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju<br />sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim<br />uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih<br />patogena poput Candida krusei, Candida parapsilosis, i Candida<br />glabrata. Na osnovu stepena i spektra antifungalne aktivnosti<br />devet izolata je odabrano za dalja istraživanja. Ispitana je<br />sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj<br />kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već<br />formirane biofilmove kandide u koncentracijama od 8 do 250<br />pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu<br />metaboličku raznolikost dobijeni su kori&scaron;ćenjem tečne<br />hromatografije visokih performansi. Tri ekstrakta sa specifičnom<br />antifungalnom aktivno&scaron;ću podvrgnuta su hemijskim analizama s<br />ciljem da se detektuju i strukturno okarakteri&scaron;u molekuli koji su<br />nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-<br />rezonantne spektroskopije otkriveno je da su aktivni<br />molekuli genistein, daidzein i staurosporin. Genistein i daidzein<br />koji su poznati fitoestrogeni poreklom iz sojinog bra&scaron;na za koje je<br />poznato da inhibiraju ključne enzime u biosintetskom putu<br />steroida. Njihovo prisustvo je u ovom istraživanju detektovano<br />usled kori&scaron;ćenja sojinog bra&scaron;na u hranljivoj podlozi. Kako<br />streptomicete u čijim ekstraktima su detektovani ovi molekuli<br />pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz<br />kompleksne hranljive podloge, mogu se uzeti u razmatranje za<br />biotehnolo&scaron;ku proizvodnju fitoestrogena. Staurosporin je<br />detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja<br />Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i<br />njegovi derivati i analozi se koriste u kao antitumorski agensi.<br />Biosinteza ovog molekula je optimizovana do prinosa od 36,94<br />mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži<br />glukozu, skrob, manitol i sojino bra&scaron;no (JS). Dalja optimizacija<br />hranljive podloge za biosintezu staurosporina ukazala je na<br />sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,<br />21,46 g/l manitola, 17,32 g/l sojinog bra&scaron;na. Primenom<br />definisanih optimalnih vrednosti i kori&scaron;ćenjem odgovarajućih<br />matematičkih modela, predviđeno je da će se na ovaj način<br />postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml<br />biomase. Validnost predviđenih rezultata potvrđena je<br />izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).<br />Ispitana je kinetika biosinteze staurosporina i produkcije biomase,<br />kao i potro&scaron;nje izvora ugljenika i razvijeni su odgovarajući<br />procesni modeli. Dodatna optimizacija je podrazumevala dodatak<br />suplemenata koji prema literature stimuli&scaron;u sekundarni<br />metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil<br />oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH<br />vrednosti (6,5, 7,5 i 8,5) a uspe&scaron;nost bioprocesa je procenjivana 7.,<br />10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka<br />da dodatak soli gvožđa značajno pospe&scaron;uje biosintezu<br />staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati<br />potvrđuju da su rizosfere medicinski značajnih biljaka značajan<br />izvor streptomiceta koje proizvode komponente sa<br />antifungalnom aktivno&scaron;ću. Izolacija novog proizvođača<br />staurosporina i optimizacija procesa njegove biosinteze<br />omogućiće dalja istraživanja ovog jedinjenja koje može biti<br />osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju<br />angiogenezu. Rezultati dobijeni u okviru ovih istraživanja<br />predstavljaju početni korak ka potencijalnoj industrijalizaciji<br />proizvodnje staurosporina.</p> / <p>Different soils are still a source of remarkable microbial diversity<br />which also reflects in the unexplored chemical diversity. Recent<br />advances in assessment of microbial diversity from soil have<br />revealed the extraordinarily rich biosynthetic potential for the<br />production of new natural products among different microbial<br />strains, especially within the group of Actinobacteria. Among<br />bacterial soil isolates, representatives of Streptomyces genus are<br />the most prolific producers of bioactive compounds. One of the<br />objectives of the present study was to isolate Streptomyces spp.<br />from the rhizosphere soils of three ethno-medicinal plants<br />collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and<br />Urtica dioica) and to screen their antifungal activity against<br />Candida spp. Morphologically different sporulating isolates (103<br />in total) were collected from rhizosphere soil samples and<br />determined as Streptomyces spp. Two different media and two<br />extraction procedures were used to induce the production and<br />facilitate identification of antifungals. Overall, 412 crude cell<br />extracts were tested against Candida albicans using disk<br />diffusion assays, with 42% (43/103) of the strains showing the<br />ability to produce antifungal agents. Also, extracts inhibited<br />growth of other important human pathogens: Candida krusei,<br />Candida parapsilosis, and Candida glabrata. Based on the<br />established degree and range of antifungal activity, nine isolates<br />were selected for further testing. Their ability to inhibit Candida<br />growth in liquid culture, to inhibit biofilm formation, and to<br />disperse pre-formed biofilms was assessed with active<br />concentrations from 8 to 250 pg/ml. High-performance liquid<br />chromatographic profiles of extracts derived from selected<br />strains were recorded, revealing moderate metabolic diversity.<br />The most potent extracts were subjected to comprehensive<br />identification and structural characterization of antifungal<br />compounds. Applying a bioactivity-guided isolation approach,<br />active compounds of three extracts were separated, and based<br />on NMR structure elucidation it was shown that active<br />compounds were genistein, daidzein and staurosporine.<br />Genistein and daidzein, soy phytoestrogens, are known to inhibit<br />key enzymes in the steroid metabolism pathway and were<br />coming from the fermentation medium containing soy flower.<br />Since isolated Streptomyces spp. showed good ability to extract<br />these molecules from complex medium, they can be further<br />considered for biotechnological production of these<br />phytoestrogens. One of the isolates, Streptomyces sp. BV410,<br />was characterized as an efficient staurosporine producer.<br />Staurosporine is a potent inhibitor of protein kinases and is<br />considered in anticancer therapy. The biotechnological<br />production of staurosporine by strain BV410 was optimized to<br />yield 36.94 mg/l after 14 days of incubation in soy flowerglucose-<br />starch-mannitol based fermentation medium (JS).<br />Further optimization of medium for biosynthesis of<br />staurosporine indicated the following optimal values of the<br />examined factors: the content of glucose of 20 g/l, starch 0.36<br />g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the<br />defined optimal values and using the appropriate mathematical<br />models, the following responses were predicted: concentration<br />of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The<br />validity of the results was confirmed by performing the<br />biosynthesis of the staurosporine in the medium with optimal<br />composition (JSSta). Kinetics of staurosporine and biomass<br />production and carbon source consumption were examined and<br />process models were developed. Additionally, optimization of<br />staurosporine production was performed with different<br />supplements which, according to literature data, had stimulative<br />effect on secondary metabolism (Zn, Fe and P salts, methyl<br />oleate, grape seed oil). In order to improve the production of<br />staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time<br />(7, 10 and 14 days) were also examined. It was found that<br />addition of FeS04 significantly improved the staurosporine yield<br />in comparison to the starting conditions (increase of 25%). Our<br />results proved that rhizosphere soils of ethno-medicinal plants<br />are a prolific source of streptomycetes, producers of compounds<br />with good antifungal activity. Isolation of the new staurosporine<br />producing strain, allowed for its detailed bioactivity assessment.<br />Staurosporine scaffold might serve as a lead structure for the<br />development of new antifungal and antiangiogenic agents. Also,<br />results obtained within this research represent the basis for the<br />further scale-up and potential industrialization of the proposed<br />production process.</p>

Much success was already achieved for the development of efficient oxyfunctionalization bioprocesses by the application of oxygenases in heterotrophic whole-cell host systems. However, several restrictions such as the technically limited O2 supply and carbohydrate-based electron supply still limit their implementation on an industrial scale concerning production rates and costs. The use of phototrophic organisms as whole-cell biocatalysts for oxygenase-based biotransformations provides an alternative and promising technology for the eco-efficient production of oxyfunctionalized value-added chemicals. While numerous cyanobacterial or microalgal bioprocesses were already developed for CO2-derived fermentations, biotransformation processes relying on the generation of activated reduction equivalents as well as O2-derived from photosynthetic water oxidation are rare. In this context, research mainly focuses on the demonstration of engineered catalysts with emphasis on the production of hydrogen. Yet, an integrated bioprocess design for the application of phototrophic organisms in redox biotransformations beyond the proof-of-concept catalyst development is lacking. This thesis aims at the integrated application of biotechnological methods and strategies for the development of eco-efficient photosynthesis-driven oxyfunctionalization processes. The main research question combines the conceptual evaluation of photosynthetic electron and O2 supply with the technical applicability of cyanobacteria as phototrophic host organisms in a hydrocarbon oxyfunctionalization bioprocess. Using a guide of integrated bioprocess design, biocatalyst, reaction, and process engineering tools are applied for the establishment of new, photosynthesis-driven bioprocesses.

No description available.

The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work. / QC 20111123 / Vinnova: BIO-AMINES / SIDA Vietnam: Production of viral proteins for vaccine development

Producing pharmaceuticals in Escherichia coli inevitably comes with an extensive purification process. This is because many of the native proteins of E. coli are immunogenic to humans, especially the heat and pH resistant endotoxins located in the membrane of E. coli. These native proteins drive up the cost of the purification, which led to a request from the biopharmaceutical company Affibody AB. They want a review on the possibilities of producing their unique Affibody®-molecules in a new, less problematic host cell. Based on a previous bachelor project, Affibody AB chose Pichia pastoris as the candidate. P. pastoris is a methylotrophic yeast that is increasing in use when it comes to producing pharmaceuticals. In this review, multiple ways of utilizing P. pastoris are presented. The process proposals are based on 4 different promoters, pAOX1, pAOX2, pFLD1 and the pGAP. The AOX1- and AOX2-promoters and the FLD1-promoter are inducible promoters that require an inducer-molecule. An inducible promoter presents the best control of the process. The GAP-promoter is a constitutive promoter, meaning that the gene is expressed continuously. A constitutive promoter provides a process which requires fewer steps and ingredients. If the Affibody®-molecules were to be produced with P. pastoris as the host cell, the products would contain less immunogenic substances. Further, P. pastoris is also a very effective option when it comes to producing protein extracellularly. This would ultimately lead to a purification process that requires less resources.

Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield. In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications. / <p>QC 20130506</p>

A big challenge within pulp and paper mills is the large quantities of waste activated sludge (WAS) that is produced during the wastewater treatment. The WAS is made up of biological cells and extra polymeric substances (EPS) and can bind a large amount of water causing difficulties to dewater the WAS. This study aimed to determine how to improve the dewatering properties of the WAS by using hydrolysis. Hydrolysis will cause the cells to disrupt and the bound water in the cells and the water trapped by the EPS can be released. Specifically, this study investigated what impact hydrolysis with heat, alkalis, and acids had on the WAS dewatering properties. In addition to the impact on the dewatering properties, the release of organic material and nutrients from the cells has also been important for biomethane production. In this study, it was specifically NH4-N, PO43- and COD that have been studied. WAS from paper mills have in general poor methane potential so it was of interest to see how the WAS was affected by hydrolysis and how hydrolysis could improve the methane production. To test the hypothesis of whether hydrolysis could affect the WAS and improve the dewatering properties, several experiments were performed. The experiments included thermal hydrolysis at temperatures of 70-90 °C, acidic hydrolysis with acids such as spent acid and acid water, and alkalis such as green liquor sludge and EOP. All acids and alkalis used in the study were chemicals that exist at the paper mills included in this study. To test the dewatering properties, methods such as TS analysis on the accept, CST-analysis, and a belt press were used. Analyses were also performed on the reject to measure the suspended solids and the nutrients NH4 – N, PO43– and COD in the WAS. This study did also determine what effect hydrolysed WAS had on the biomethane potential. In this study were the paper mills BillerudKorsnäs in Skärblacka and SCA in Östrand included. Hence was sludge from the two mills of interest to analyse. This study has shown better dewatering properties with an increase in the total solids (in the accept) after the thermal hydrolysis, the acidic hydrolysis with spent acid, and the alkali hydrolysis with green liquor sludge. Specifically did the acidic hydrolysis with spent acid improve the dewatering properties in terms of an increase in TS in %. The biggest increase in TS in % could be seen after using 10% spent acid ratio. The TS for the WAS from SCA Östrand increased in this experiment by 107 %. The thermal hydrolysis also showed promising results both in terms of dewatering properties and in the release of organic material. The biochemical methane potential test results showed a better and more rapid stabilized production of biomethane after hydrolysis of WAS compared to untreated WAS. The thermal hydrolysis both increased the rate of production and the total amount of methane produced. The thermally hydrolysed WAS from SCA Östrand improved the methane production from 77 Nml methane/g VS to 95 Nml methane/ g VS. The WAS from BillerudKorsnäs improved the methane production from 40 Nml methane/ g VS to 55 Nml methane/ g VS. These results, both from the methane potential tests and the results of the increased dewatering properties, show that the concept with hydrolysing should be evaluated further for improving the dewatering of the WAS.

Les dernières années ont permis des développements très rapides, tant au niveau de l’élaboration que de l’application, d’algorithmes de commande prédictive non linéaire (CPNL), avec une gamme relativement large de réalisations industrielles. Un des obstacles les plus significatifs rencontré lors du développement de cette commande est lié aux incertitudes sur le modèle du système. Dans ce contexte, l’objectif principal de cette thèse est la conception de lois de commande prédictives non linéaires robustes vis-à-vis des incertitudes sur le modèle. Classiquement, cette synthèse peut s’obtenir via la résolution d’un problème d’optimisation min-max. L’idée est alors de minimiser l’erreur de suivi de la trajectoire optimale pour la pire réalisation d'incertitudes possible. Cependant, cette formulation de la commande prédictive robuste induit une complexité qui peut être élevée ainsi qu’une charge de calcul importante, notamment dans le cas de systèmes multivariables, avec un nombre de paramètres incertains élevé. Pour y remédier, une approche proposée dans ces travaux consiste à simplifier le problème d’optimisation min-max, via l’analyse de sensibilité du modèle vis-à-vis de ses paramètres afin d’en réduire le temps de calcul. Dans un premier temps, le critère est linéarisé autour des valeurs nominales des paramètres du modèle. Les variables d’optimisation sont soit les commandes du système soit l’incrément de commande sur l’horizon temporel. Le problème d’optimisation initial est alors transformé soit en un problème convexe, soit en un problème de minimisation unidimensionnel, en fonction des contraintes imposées sur les états et les commandes. Une analyse de la stabilité du système en boucle fermée est également proposée. En dernier lieu, une structure de commande hiérarchisée combinant la commande prédictive robuste linéarisée et une commande par mode glissant intégral est développée afin d’éliminer toute erreur statique en suivi de trajectoire de référence. L'ensemble des stratégies proposées est appliqué à deux cas d'études de commande de bioréacteurs de culture de microorganismes. / The last few years have led to very rapid developments, both in the formulation and the application of Nonlinear Model Predictive Control (NMPC) algorithms, with a relatively wide range of industrial achievements. One of the most significant challenges encountered during the development of this control law is due to uncertainties in the model of the system. In this context, the thesis addresses the design of NMPC control laws robust towards model uncertainties. Usually, the above design can be achieved through solving a min-max optimization problem. In this case, the idea is to minimize the tracking error for the worst possible uncertainty realization. However, this robust approach tends to become too complex to be solved numerically online, especially in the case of multivariable systems with a large number of uncertain parameters. To address this shortfall, the proposed approach consists in simplifying the min-max optimization problem through a sensitivity analysis of the model with respect to its parameters, in order to reduce the calculation time. First, the criterion is linearized around the model parameters nominal values. The optimization variables are either the system control inputs or the control increments over the prediction horizon. The initial optimization problem is then converted either into a convex optimization problem, or a one-dimensional minimization problem, depending on the nature of the constraints on the states and commands. The stability analysis of the closed-loop system is also addressed. Finally, a hierarchical control strategy is developed, that combines a robust model predictive control law with an integral sliding mode controller, in order to cancel any tracking error. The proposed approaches are applied through two case studies to the control of microorganisms culture in bioreactors.

La conception de souches efficacespour la production de composés d’intérêt offredes potentiels immenses qui restent trop peu exploitéspar manque de lien entre les étapes d’optimisationde la conception de souche et celle dela conduite du bioprocédé.Pour combler ce manque, cette thèse proposeune description des bioprocédés intégrant pleinementle fonctionnement interne des microorganismesimpliqués dans la production decomposés d’intérêt. Cette description permetd’optimiser simultanément la souche et le procédépour maximiser la production d’un composéd’intérêt en respectant les contraintes attachéesà ces deux étapes.Dans un premier temps, une nouvelle classe demodélisation de bioprocédés est développée, àl’interface entre les modèles intracellulaires degestion de ressources et les modèles macroscopiquesusuellement utilisés dans la commandede bioprocédés en bioréacteurs. Dans un secondtemps, des contraintes liées à l’implémentationbiologique de la stratégie de contrôle sont intégréesau problème. Ceci permet d’obtenir uneconception plus réaliste du point de vue de l’ingénieriedes génomes. Enfin, la dernière partiede la thèse montre que la méthodologie présentéejusqu’alors sur un modèle agrégé peut êtreétendue à des représentations détaillées du comportementdes micro-organismes. / The design of efficient strains forthe production of compounds of interest offerstremendous potentials that remain insufficientlyexploited due to the lack of link between theoptimization stages of strain design and that ofbioprocess control.This thesis proposes a description of bioprocessesthat fully integrates the internal functioningof micro-organisms involved in the productionof compounds of interest. This descriptionallows the strain and process to be optimizedsimultaneously to maximize the productionof a compound of interest while respecting theconstraints attached to these two stages.First, a new bioprocess modelling class is developedat the interface between intracellular resourceallocation models and macroscopic modelscommonly used in bioprocess control. In asecond stage, constraints linked to the biologicalimplementation of the control strategy are integratedinto the problem. This provides a morerealistic genome engineering design. Finally, thelast part of the thesis shows that the methodologypresented so far on an aggregate model canbe extended to detailed representations of thebehaviour of micro-organisms.

Plant derived natural products represent some of the most effective anti-cancer and anti-infectious disease pharmaceuticals available today. However, uncertainty regarding the feasibility of commercial supply due to the limited availability of many plants in nature has resulted in a dramatic reduction in the use of natural products as leads in modern drug discovery. Plant cell suspension culture, consisting of dedifferentiated plant cells grown in vitro and amenable to large scale industrial biotechnology processes, is a production alternative which promises renewable and economical supply of these important drugs. The widespread application of this technology is limited by low product yields, slow growth rates, challenges in scale-up, and above all, variability in these properties, which is poorly understood. Plant cells grow as aggregates in suspension cultures ranging from two to thousands of cells (less than 100 micron to well over 2 mm). Aggregates have long been identified as an important feature of plant cell culture systems, as they create microenvironments for individual cells with respect to nutrient limitations, cell-cell signaling, and applied shear in the in vitro environment. Despite its purported significance, a rigorous engineering analysis of aggregation has remained elusive. In this thesis, aggregation was characterized, analyzed, and modeled in Taxus suspension cultures, which produce the anti-cancer drug paclitaxel. A technique was developed to reliably and routinely measure aggregate size using a Coulter counter. The analysis of aggregate size as a process variable was then used to evaluate the effect of aggregation on process performance, and the analysis of single cells isolated from different sized aggregates was used to understand the effect of aggregation on cellular metabolism and heterogeneity. Process characterization studies indicated that aggregate size changed over a batch cycle as well as from batch to batch, so a population balance equation model was developed to describe and predict these changes in the aggregate size distribution. This multi-scale engineering approach towards understanding plant cell aggregation serves as an important step in the development of rational strategies aimed at controlling the process variability which has heretofore limited the application of plant cell culture technology.