This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.

e Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance for global nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing different classes of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetes phylum. We used 13 C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of all strains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyerhof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentially used the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against the oxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathway in the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifically rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tricarboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glucose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling of mono-and polysaccharides by bacterial communities in marine ecosystems. Marine bacteria influence global environmental dynamics in fundamental ways by controlling the biogeochemistry and productivity of the oceans (1). Due to their importance, marine microorganisms have been studied intensively (2). In particular, their mechanisms for metabolizing carbon and other nutrients have attracted attention, because they directly or indirectly affect the biogeochemical status of seawater (3). A prominent nutrient in seawater is glucose, the most abundant free neutral aldose (4). Current estimates of glucose concentrations in seawater indicate an almost ubiquitous distribution in the oceans in a nanomolar range (5). Particularly, large amounts of glucose are available in coastal habitats, e.g., during bloom situations (6). In fact, a large fraction (Ͼ30%) of bacterial growth can be supported by this monosaccharide in some oceans (7,8). Furthermore, glucose is the dominant component of dissolved polysaccharides, which constitute up to 15% of marine dissolved organic matter (9). The turnover of the (monomeric and polymeric) glucose pool in different oceanic regions ranges from days to months, and glucose assimilation in marine surface waters may represent up to 40% of bacterial carbon production (5). Taken together, bacteria that use glucose are common in the sea (10), and glucose is a representative model nutrient to monitor carbon uptake by heterotrophic marine bacteria (11). At this point, questions that arise from current knowledge concern...

The fed -batch technique is the most frequently applied operation mode for an effi cient biotechnological production. It is often characterized by the achievement of higher product yields compared to the batch mode. For the case when the fedbatch process is operated under substrate -limiting conditions, substrate uptake rates lower than the maximum uptake capacities of cells occur. Thus, the accumulation of branch -point intermediate compounds of the cell ' s metabolism is more or less avoided. The synthesis of unwanted side -products is not at all or only supported barely with low amounts of substrate. Usually, under substrate -limiting conditions, carbon is provided for the most important products in order to keep the cellular system functional and viable.Such substrate -limiting conditions are easily achieved at the laboratory scale and pilot scale. Most often, the feed solution is introduced through a pipe at the top of the fermenter. A mixing time of several seconds is achieved in stirred tank reactor s ( STR s), which is so fast that the even distribution of the substrate in the reactor is not disturbed. Hence, even up to high cell densities, no accumulation of unwanted byproducts under fed -batch conditions is observed, except that viscosity is very high and oxygen availability becomes insuffi cient in late process stages. However, this is different in biotechnological production in large -scale fermenters. Due to mechanical and economical limitations of the power input, the mixing time of an STR increases 10 -fold or more when reaching liquid volumes of several cubic meters [1 -3] . This leads to a faster conversion by the microorganisms near the feeding zone, while far away from it production of the substrate ceases with increasing cell concentration. The resulting gradients near the feeding point alter the process performance [4,5] . In particular, substrate excess, which thwarts the idea of a fed -batch process, can lead to the synthesis of unwanted byproducts [6] . Due to the movement of cells in the reactor, they are exposed to ongoing oscillating environmental conditions where they fl ip between substrate excess and 16 Biopharmaceutical Production Technology, First Edition. Edited by Ganapathy Subramanian. Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are

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