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In this study, the removal of IBE from aqueous solutions by gas stripping has been characterized. The effect of one or more components in the solution on the kinetics of the separation has been studied, both at 37 °C and at 70 °C. Gas stripping has been applied to batch, repeated batch and continuous cultures of Clostridium beijerinckii grown on a glucose/xylose mixed sugar substrate mimicking lignocellulosic hydrolysates, with the aim of finding optimal conditions for a stable IBE-producing culture with high productivity. An innovative repeated-batch process has been demonstrated in which the gas-stripping is performed at 70 °C, resulting in a prolonged stable IBE culture.

Biomass fermentation to Iso-propanol, Butanol and Ethanol (IBE) is particularly important as IBE is a common building block in the development of biorefineries and IBE-producing bacteria are robust industrial organisms, capable to utilize the sugars of the lignocellulosic biomass. Research is focused on increasing fermentation yields and the reduction of energy that is required to separate the volatile products. The paper addresses both of these challenges combining experimental innovations with a systems engineering approach. IBE is recovered from a gas-stripped fermenter whose potential for adsorption is researched and integrated with downstream options for separation. Design and integration is assisted using a systems approach that relies on mathematical models that regress and extrapolate experimental data for scale-up calculations. Process integration involves synthesis challenges to define biorefinery portfolios and systems integration to combine fermentation, stripping, adsorption, and distillation. The analysis considers 4 alternative biorefinery cases and presents results with significant savings in energy use and costs (up to 87% savings reported) after the application of energy integration to the IBE plant. Scenarios are analysed economically and confirm benefits in the use of adsorption and viable production yields.

Hydrogen was produced from carrot pulp hydrolysate, untreated carrot pulp and (mixtures of) glucose and fructose by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana in pH-controlled bioreactors. Carrot pulp hydrolysate was obtained after enzymatic hydrolysis of the polysaccharide fraction in carrot pulp. The main sugars in the hydrolysate were glucose, fructose, and sucrose. In fermentations with glucose hydrogen yields and productivities were similar for both strains. With fructose the hydrogen yield of C. saccharolyticus was reduced which might be related to uptake of glucose and fructose by different types of transport systems. With T. neapolitana the fructose consumption rate and consequently the hydrogen productivity were low. The hydrogen yields of both thermophiles were 2.7–2.8 mol H2/mol hexose with 10 g/L sugars from carrot pulp hydrolysate. With 20 g/L sugars the yield of T. neapolitana was 2.4 mol H2/mol hexose while the yield of C. saccharolyticus was reduced to 1.3 mol H2/mol hexose due to high lactate production in the stationary growth phase. C. saccharolyticus was able to grow on carrot pulp and utilized soluble sugars and, after adaptation, pectin and some (hemi)cellulose. No growth was observed with T. neapolitana when using carrot pulp in agitated fermentations. Enzymatic hydrolysis of the polysaccharide fraction prior to fermentation increased the hydrogen yield with almost 10% to 2.3 g/kg of hydrolyzed carrot pulp.

Production of hydrogen by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana was studied in serum flasks and in pH-controlled bioreactors with glucose, and hydrolyzed and untreated potato steam peels (PSP) as carbon sources. Two types of PSP hydrolysates were used: one in which the starch in the PSP was liquefied with alpha-amylase, and one in which the liquefied starch was further hydrolyzed to glucose by amyloglucosidase. When the PSP hydrolysates or untreated PSP were added at circa 10–14 g/L of glucose units, both strains grew well and produced hydrogen with reasonable to high molar yields (2.4–3.8 moles H2/mole glucose units), and no significant production of lactate. The hydrogen production rates and yields were similar with untreated PSP, hydrolyzed PSP, and pure glucose, showing that C. saccharolyticus and T. neapolitana are well equipped for the utilization of starch. When the concentrations of the substrates were increased, growth and hydrogen production of both strains were hampered. At substrate concentrations of circa 30–40 g/L of glucose units, the molar hydrogen yield of C. saccharolyticus was severely reduced due to the formation of high amounts of lactate, while T. neapolitana was unable to grow at all. The results showed that PSP and PSP hydrolysates are very suitable substrates for efficient fermentative hydrogen production at moderate substrate loadings.

The aim of this work was to evaluate the potential of employing biomass resources from different origin as feedstocks for fermentative hydrogen production. Mild-acid pretreated and hydrolysed barley straw (BS) and corn stalk (CS), hydrolysed barley grains (BG) and corn grains (CG), and sugar beet extract (SB) were comparatively evaluated for fermentative hydrogen production. Pretreatments and/or enzymatic hydrolysis led to 27, 37, 56, 74 and 45 g soluble sugars/100 g dry BS, CS, BG, CG and SB, respectively. A rapid test was applied to evaluate the fermentability of the hydrolysates and SB extract. The thermophilic bacterium Caldicellulosiruptor saccharolyticus showed high hydrogen production on hydrolysates of mild-acid pretreated BS, hydrolysates of BG and CG, and SB extract. Mild-acid pretreated CS showed limited fermentability, which was partially due to inhibitory products released in the hydrolysates, implying the need for the employment of a milder pretreatment method. The difference in the fermentability of BS and CS is in strong contrast to the similarity of the composition of these two feedstocks. The importance of performing fermentability tests to determine the suitability of a feedstock for hydrogen production was confirmed.

In measuring fructose 1,6-bisphosphatase (EC 3.1.3.11; FBPase) in potato tubers, we used anti PFP antibodies to ensure that the assay was specific for FBPase and that PPi:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90: PFP) did not contribute to the production of fructose 6-phosphate (Fru-6-P). The involvement of cytosolic FBPase in cold sweetening was determined by monitoring enzyme activity during storage ofSolanum tuberosum cv. Erntestolz tubers at 2 or 8°C. In contrast to the rapid increase in sucrose and reducing sugars, the specific activity of FBPase showed no change and PFP rose slightly during storage at 2°C. Sugar concentrations and enzyme activities remained virtually unchanged during storage at 8°C. These data allow the interconversion of fructose 1.6-bisphosphate (Fru-1,6-P2) to Fru-6-P and vice versa by FBPase and PFP, respectively, in potato tubers, but do not support a causal relationship between coarse metabolic control of FBPase and cold-induced sweetening.

The accumulation of reducing sugars, sucrose and hexose phosphates in cv. Bintje and genotype KW77-2916 during storage at 2, 4, or 8°C was studied in relation to several catalytic activities. Bintje tubers accumulated sugars during storage at 2 or 4°C, whereas KW77-2916 showed reduced cold-sweetening at 2°C. The increase in glucose 6-phosphate and sucrose occurred concurrently and preceded the increase in reducing sugar concentration. Phosphorylase activity showed a strong interaction with temperature, storage duration and sugar accumulation in both genotypes. Invertase activity increased in Bintje concomitantly with the increase in reducing sugars, but this effect was less obvious in KW77-2916. The activities of other glycolytic and Krebs cycle enzymes showed no obvious correlation with sugar accumulation. It is suggested that the increase in phosphorylase activity acts as a triggering event in the sweetening of potato tubers during cold storage.

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2 degrees C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4 degrees C. During storage at 8 degrees C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8 degrees C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8 degrees C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4 degrees C but remained the same throughout storage at 8 degrees C. After exposure to 2 degrees C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4 degrees C but decreased slightly at 8 degrees C. All observed changes became annulled after transfer of cold stored tubers to 18 degrees C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.

NMR analysis of (13)C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H(2) per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g(-1) h(-1) and 20 mmol l(-1) h(-1). An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.