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The objective of this work was to map and compare the technical suitability of different raw materials for biological hydrogen production. Our model was based on hydrogen yield potential, sugar mobilization efficiency, fermentability and coproduct yield and value. The suitability of the studied raw materials was ranked in the order: sugar beet juice > sweet sorghum juice > potato steam peels > barley straw > miscanthus > sweet sorghum bagasse > carrot press cake > wheat grains > wheat straw > wheat bran. The results indicated that raw materials with similar chemical composition may have different technical suitability for hydrogen production. Therefore, further research on the specific technical characteristics, including pretreatment requirements, of each raw material is recommended.

Biodiesel production from cotton-seed cake (CSC) and the pretreatment of the remaining biomass for dark fermentative hydrogen production was investigated. The direct conversion to biodiesel with alkali free fatty acids neutralization pretreatment and alkali transesterification resulted in a biodiesel with high esters content and physicochemical properties fulfilling the EN-standards. Blends of cotton-seed oil methyl esters (CME) and diesel showed an improvement in lubricity and cetane number. Moreover, CME showed good compatibility with commercial biodiesel additives. On the basis of conversion of the remaining CSC to sugars fermentable towards hydrogen, the optimal conditions included removal of the oil of CSC and pretreatment at 10% NaOH (w/w dry matter). The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production, 84–112% of the control, from NaOH-pretreated CSC and low hydrogen production, 15–20% of the control, from the oil-rich and not chemically pretreated CSC, and from Ca(OH)2-pretreated CSC.

Integrating of lignocellulose-based and starch-rich biomass-based hydrogen production was investigated by mixing wheat straw hydrolysate with a wheat grain hydrolysate for improved fermentation. Enzymatic pretreatment and hydrolysis of wheat grains led to a hydrolysate with a sugar concentration of 93.4 g/L, while dilute-acid pretreatment and enzymatic hydrolysis of wheat straw led to a hydrolysate with sugar concentration 23.0 g/L. Wheat grain hydrolysate was not suitable for hydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus at glucose concentrations of 10 g/L or higher, and wheat straw hydrolysate showed good fermentability at total sugar concentrations of up to 10 g/L. The mixed hydrolysates showed good fermentability at the highest tested sugar concentration of 20 g/L, with a hydrogen production of 82–97% of that of the control with pure sugars. Mixing wheat grain hydrolysate with wheat straw hydrolysate would be beneficial for fermentative hydrogen production in a biorefinery.

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.

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H3PO4 and H2SO4, and to a lesser extent, HNO3. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.

Four dilute-acid pretreated and hydrolysed lignocellulosic raw materials were evaluated as substrates for fermentative hydrogen production by Caldicellulosiruptor saccharolyticus. Their fermentability was ranked in the order: barley straw > wheat straw > corn stalk > corn cob. The content of 5-hydroxymethylfurfural (HMF) in medium with hydrolysates prepared from corn cob (1.0 g/L) and corn stalk (0.8 g/L), respectively, reached levels likely to be toxic for growth of C. saccharolyticus. HMF was absent in wheat and barley straw hydrolysates. Furfural concentrations in media with wheat straw, barley straw and corn stalk hydrolysates were low (0.2-0.3 g/L), while it was higher in corn cob hydrolysates (0.6 g/L). (Less)

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.

Pretreatment of sweet sorghum bagasse, an energy crop residue, with NaOH for the production of fermentable substrates, was investigated. Optimal conditions for the alkaline pretreatment of sweet sorghum bagasse were realized at 10% NaOH (w/w dry matter). A delignification of 46% was then observed, and improved significantly the efficiency of enzymatic hydrolysis. Under hydrolysis conditions without pH control, up to 50% and 41% of the cellulose and hemicellulose contained in NaOH-pretreated sweet sorghum bagasse were converted by 24 h enzymatic hydrolysis to soluble monomeric sugars. The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed normal growth on hydrolysates of NaOH-pretreated biomass up to a sugar concentration of 20 g/L. Besides hydrogen, the main metabolic products detected in the fermentations were acetic and lactic acid. The maximal hydrogen yield observed in batch experiments under controlled conditions was 2.6 mol/mol C6 sugar. The maximal volumetric hydrogen production rate ranged from 10.2 to 10.6 mmol/(L h). At higher substrate concentrations the production of lactic acid increased at the expense of hydrogen production.

Hydrogen can be produced through dark anaerobic fermentation using carbohydrate-rich biomass, and through photofermentation using the organic acids produced from dark fermentation. Sugar beet is an ideal energy crop for fermentative production of hydrogen in the EU due to its environmental profile and its potential availability in the area. In this work, various aspects of cultivating sugar beet in the EU for biohydrogen were highlighted, with special focus on The Netherlands and Greece. Moreover, fermentation of sugar beet juice with Caldicellulosiruptor saccharolyticus at sucrose concentration 10 g/l was performed, and was found comparable to the fermentation on pure sucrose except that the hydrogen production was 10% higher on sugar beet juice. A conservative estimate of the annual hydrogen potential in the EU was made (300 × 106 kg hydrogen), considering the utilization of sugar beet pulp in hydrogen production.

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup− mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH4+ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H2/mol sucrose) and maximum volumetric productivity (7.1 mmol H2/Lc.h) were obtained in the absence of NH4+. The effluent of the dark fermentation containing no NH4+ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H2/Lc.h) were attained using the hup− mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H2/mol sucrose in dark fermentation to 13.7 mol H2/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H2/mole of sucrose) by sequential dark and photofermentation.

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.

Stripping gas is generally used to improve hydrogen yields in fermentations. Since CO2 is relatively easy to separate from hydrogen it could be an interesting stripping gas. However, a higher partial CO2 pressure is accompanied with an increased CO2 uptake in the liquid, where it hydrolyses and induces an increased requirement of NaOH to maintain the pH. This enhances the osmotic pressure in the culture by 30%, which inhibited the growth of Caldicellulosiruptor saccharolyticus. indications for this conclusion are: i) inhibition could almost completely be circumvented by reducing the bicarbonate through decreasing the pH (from 6.5 to 5.5), ii) Growth rates were reduced by 60 +/- 10% at an osmotic pressure of 0.218 +/- 0.005 osm/kg H2O independently of the stripping gas, iii) Increased extracellular DNA and protein concentrations were observed as a function of the osmotic pressure. In addition to growth inhibition, the increased sodium bicarbonate in the effluent will contribute to a negative environmental impact when applied at industrial scale. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All lights reserved. (Less)

Non-axenic operation of a 400 L trickle bed reactor inoculated with the thermophile Caldicellulosiruptor saccharolyticus, yielded 2.8 molH 2mol hexose converted. The reactor was fed with a complex medium with sucrose as the main substrate, continuously flushed with nitrogen gas, and operated at 73°C. The volumetric productivity was 22 mmolH2(L filterbed h). Acetic acid and lactic acid were the main by-products in the liquid phase. Production of lactic acid occurred when hydrogen partial pressure was elevated above 2% and during suboptimal fermentation conditions that also resulted in the presence of monoand disaccharides in the effluent. Methane production was negligible. The microbial community was analyzed at two different time points during operation. Initially, other species related to members of the genera Thermoanaerobacterium and Caldicellulosiruptor were present in the reactor. However, these were out-competed by C. saccharolyticus during a period when sucrose was completely used and no saccharides were discharged with the effluent. In general, the use of pure cultures in non-sterile industrial applications is known to be less useful because of contamination. However, our results show that the applied fermentation conditions resulted in a culture of a single dominant organism with excellent hydrogen production characteristics. © 2008 Wiley Periodicals, Inc.

Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of β-1,3- and β-1,4-linked β- d -glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli , and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum . Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum , were cloned separately into C. beijerinckii . To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 ( celA ) and pWUR4 ( celD ). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD ). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.

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.

Biological hydrogen production by a sequential operation of dark and photofermentation is a promising route to produce hydrogen. The possibility of using renewable resources, like biomass and agro-industrial wastes, provides a dual effect of sustainability in biohydrogen production and simultaneous waste removal. In this study, photofermentative hydrogen production on effluents of thermophilic dark fermentations on glucose, potato steam peels (PSP) hydrolysate and molasses was investigated in indoor, batch operated bioreactors. An extreme thermophile Caldicellulosiruptor saccharolyticus was used in the dark fermentation step, and Rhodobacter capsulatus (DSM1710) was used in the photofermentation step. Addition of buffer, Fe and Mo to dark fermentor effluents (DFEs) improved the overall efficiency of hydrogen production. The initial acetate concentration in the DFE needed to be adjusted to 30–40 mM by dilution to increase the yield of hydrogen in batch light-supported fermentations. The thermophilic DFEs are suitable for photofermentative hydrogen production, provided that they are supplemented with buffer and nutrients. The overall hydrogen yield of the two-step fermentations was higher than the yield of single step dark fermentations.

Microtubers of potato cultivar Kennebec were stored for two months at 2 or 8°C and sampled at selected intervals for the determination of glucose, fructose, sucrose and sprout weight. The aim of this experiment was to determine whether these microtubers show the same response to storage temperature with respect to sweetening as field grown tubers. During storage at 2 °C, reducing sugars and sucrose increased rapidly to a concentration of over 1.0 and 0.7 g/100 g fresh weight, respectively. In contrast, in tubers kept at 8°C, only a relatively slight increase in reducing sugar concentration to 0.3 g/100 g fresh weight was observed and sucrose concentration remained virtually the same.

Dealing specifically with liquid and gaseous biofuels that can be produced from renewable resources using different processes, Biofuels provides a broad overview of biofuel developments from both a technical angle and an economical angle. Trends on prices, markets, and growth are discussed in depth. The link between the technical and economical developments is clearly highlighted throughout the entire book

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.

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans . CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli . The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli -produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H2 in the gas phase ( partial hydrogen pressure (pH2) of (1-2) · 104 Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H2 ( pH2 of 5.6 · 104 Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70°C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 255-262, 2003. (Less)

Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and beta-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium.

SUMMARY: In cell-free extracts of Thiobacillus versutus, an organism which has been reported to be isocitrate lyase negative, an isocitrate lyase activity of 52 ± 18 nmol min−1 (mg protein)−1 was observed after anaerobic growth in a chemostat on acetate plus nitrate, i.e. during denitrification. Following growth on succinate plus nitrate, isocitrate lyase activity was only 1 ± 2 nmol min−1 (mg protein)−1. In cell-free extracts derived from aerobic chemostat cultures isocitrate lyase activity was always nil. The identity of the enzyme was analysed using a number of different methods, namely (a) three different enzyme assays, (b) 13C-NMR spectroscopy of the reaction products, (c) HPLC analysis of the reaction products, (d) mass spectrometry of derivatized glyoxylate enzymically produced from isocitrate and (e) radiography of derivatized glyoxylate enzymically produced from [14C]citrate. All these methods gave results consistent with the enzyme-catalysed conversion of isocitrate to glyoxylate and succinate.

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min-1·mg-1 protein in thiosulphate-grown cells to 58 nmol·min-1·mg-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.