Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621

Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K_m 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K_m 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu²⁺ and Pb²⁺ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry.     

Copper sulfide precipitation by yeasts from Acid mine-waters

Two strains of Rhodotorula and one of Trichosporon precipitated dissolved copper with H₂S formed by reducing elemental sulfur with glucose. Iron stimulated this activity under certain conditions. In the case of Rhodotorula strain L, iron stimulated copper precipitation aerobically at a copper concentration of 18 but not 180 μg/ml. Anaerobically, the L strain required iron for precipitation of copper from a medium with 180 μg of copper per ml. Rhodotorula strain L was able to precipitate about five times as much copper anaerobically as aerobically. The precipitated copper was identified as copper sulfide, but its exact composition could not be ascertained. Iron was not precipitated by the H₂S formed by any of the yeasts. Added as ferric iron, it was able to redissolve copper sulfide formed aerobically by Rhodotorula strain L from 18 but not 180 μg of copper per ml of medium. Since the yeasts were derived from acid mine-waters, their ability to precipitate copper may be of geomicrobial importance.     

Boswellia serrata Preserves Intestinal Epithelial Barrier from Oxidative and Inflammatory Damage

Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD), however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM) use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H₂O₂ or INF-γ+TNF-α, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER) and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin) immunofluorescence. The expression of phosphorylated NF-κB and reactive oxygen species (ROS) generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE) and its pure derivative acetyl-11-keto-β-boswellic acid (AKBA), were tested at 0.1-10 μg/ml and 0.027μg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H₂O₂ or INF-γ+TNF-α treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-κB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H₂O₂ exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study elucidates the pharmacological mechanisms mediated by BSE, in protecting intestinal epithelial barrier from inflammatory damage and supports its use as safe adjuvant in patients affected by IBD.     

General Biochemical Characterization of Thermostable Pullulanase and Glucoamylase from Clostridium thermohydrosulfuricum

Cell extracts of Clostridium thermohydrosulfuricum, an anaerobic bacterium which ferments starch into ethanol at 65°C, contained both pullulanase and glucoamylase activities. The general physiochemical and catalytic properties of these enzyme activities were compared. Pullulanase and glucoamylase activities were stable and optimally active at 85 and 75°C, respectively. The pH optima for activity and pH stability ranges were, respectively, 5.5 to 6 and 4.5 to 5.5 for pullulanase and 4 to 6 and 5 to 6 for glucoamylase. The apparent [S]_0.5v and V_max for pullulanase activity on pullulan were 0.33 mg/ml and 2.6 U/mg of protein. The apparent [S]_0.5v and V_max for glucoamylase activity on starch were of 0.41 mg/ml and 0.31 U/mg of protein. These enzymes were active and stable in the presence of air or 10% (vol/vol) ethanol. These enzyme activities allowed the organism to actively degrade raw starch into glucose in the absence of significant α-amylase activity.     

H<Subscript>2</Subscript>-Producing bacterial communities from a heat-treated soil inoculum

Hydrogen gas (60% H₂) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l^–1). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H₂ by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30°C and 37°C). At 30-h HRT, the H₂ production rate was 80 ml h^–1 and yield was 0.91 mol H₂/mol glucose. At 10-h HRT, the H₂ production rate was more than 5 times higher at 436 ml h^–1, and yield was 1.61 mol H₂/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30°C to 37°C, the steady-state H₂ production rate increased slightly to 463 ml h^–1 and yield was 1.8 mol H₂/mol glucose. Compared to 30°C, RISA fingerprints at 37°C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).     

Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209 ml H₂/l medium, 1.97 mol H₂/mol glucose and 104.20 ml H₂/g CDW h⁻¹ were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H₂, and is another model microorganism for biohydrogen investigations.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

Metabolic precursors of surfactant disaturated-phosphatidylcholine in preterms with respiratory distress

Our objective was to study the metabolic precursors of surfactant disaturated-phosphatidylcholine (DSPC) in preterm infants with respiratory distress syndrome (RDS) on mechanical ventilation. We performed 46 DSPC kinetic studies in 23 preterms on fat-free parenteral nutrition and mechanical ventilation (birth weight = 1167 ± 451 g, gestational age = 28.5 ± 2.0 weeks). Eight infants received a simultaneous intravenous infusion of U¹³C-glucose and [16,16,16]²H-palmitate, eight infants received U¹³C-glucose and ²H₂O, and seven received U¹³C-palmitate and ²H₂O. Surfactant DSPC kinetics were calculated from the isotopic enrichments of DSPC-palmitate from sequential tracheal aspirates and its metabolic precursors in plasma or urine. DSPC fractional synthesis rate (FSR) was 17 ± 11, 21 ± 16, and 15 ± 6%/day from glucose, palmitate, and body water, respectively (P = 0.36). DSPC-FSR from U¹³C-glucose and ²H₂O were significantly correlated and yielded similar estimates (difference of –0.1 ± 3%) (P = 0.91). The difference in the 15 infants receiving palmitate versus ²H₂O or palmitate versus glucose was +6.0 ± 12%/day (P = 0.21). There was a significant correlation between DSPC-FSRs from plasma glucose and plasma FFA. The contribution of glucose versus palmitate to DSPC-FSR was 49 ± 20% versus 51 ± 20%, respectively. Plasma glucose and FFA showed similar contributions to DSPC-FSR in infants with RDS and fat-free parenteral nutrition. FSRs from ²H₂O or glucose were highly correlated.     

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree,Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L‐tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L‐tryptophan. The preferred carbon and nitrogen sources were glucose and KNO₃ and the optimum concentrations 1% and 0.02%, respectively. FeSO₄ × 7 H₂O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO₄ × 7 H₂O (10 μg/ml), KNO₃ (0.02%) as well as EDTA (5 μg/ml) and L‐tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree‐Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.     

Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background

Pure bacterial strains give better yields when producing H₂ than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂ production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.

Results

Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20?L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂ production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂ yields were obtained for starch fermentations, and the highest yield of 2.91?mol?H₂/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5?L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂ at higher rates than the pure Clostridium cultures, without negatively affecting the H₂ yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂ during 13?days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.

Conclusions

The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H₂ using glucose and starch as carbon sources. The artificial co-cultures produced H₂ at higher rates than the pure strains, while the H₂ yields were only slightly affected.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

In vitro cultivation of Hymenolepis diminuta: effect of antibiotics on the growth of three-day-old worms removed from the rat

The effects of several antibacterial or antifungal antibiotics on the growth of 3-day-old Hymenolepis diminuta cultivated in vitro were investigated. Worms were recovered from the rat and cultured in roller tubes for 4 days without a medium change. The medium contained horse serum, yeast extract, and liver extract; the gas phase was 5% CO₂ in N₂. It was found that penicillin and streptomycin did not inhibit the growth of worms at concentrations lower than 5000 units and μg/ml of medium, respectively. Cycloheximide was toxic to H. diminuta, retarding or inhibiting growth at levels higher than 1.5 μg/ml. The antifungal antibiotics nystatin and amphotericin B did not affect worm growth at concentrations lower than 1000 units and 123 μ/ml of medium, respectively.The use of penicillin and streptomycin, and nystatin or amphotericin B can be recommended for the control of bacterial and fungal contaminants in the cultivation of H. diminuta removed from the rat.     

Cloning and overexpression of raw starch digesting α-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified recombinant α-amylase (AmyBS-I) in raw starch digestion and baking industry

Considering the economic and industrial relevance of α-amylases used in food and starch industries, a raw starch digesting α-amylase gene (amyBS-I) from Bacillus subtilis strain AS01a was cloned and expressed in Escherichia coli BL21 cells. The gene also includes its signal peptide sequence (SPS) for facilitating the efficient extracellular expression of recombinant α-amylase (AmyBS-I) in correctly folded (enzymatically active) form. The native AmyBS-I consists of 659 amino acids with a molecular mass and pI of 72,387 Da and 5.8, respectively. The extracellular secretion of AmyBS-I after response surface optimization of culture conditions was found to be 7-fold higher as compared to its production under non-optimized conditions. Purified AmyBS-I demonstrated optimum activity at 70 °C and pH 6.0. It shows K_m and V_max values toward soluble starch as 2.7 mg/ml and 454 U/ml, respectively. Further, it does not require Ca²⁺ ion for its α-amylase activity/thermo-stability, which is an added advantage for its use in the starch industry. The AmyBS-I also hydrolyzed a wide variety of raw starches and produced maltose and glucose as main hydrolyzed products. The bread dough supplemented with AmyBS-I showed better amelioration of the bread quality as compared to the bread supplemented with commercial α-amylase.     

Sufficient intake of high amylose cornstarch maintains high colonic hydrogen production for 24 h in rats

Colonic hydrogen (H₂) can suppress oxidative stress and damage in the body. We examined the minimum requirement of high amylose cornstarch (HAS) to maintain high colonic H₂ production for 24 h. Ileorectostomized and sham-operated rats were fed a control diet supplemented with or without 20% HAS for 7 days. Colonic starch utilization was determined. Next, rats were fed the control diet with or without 10% or 20% HAS for 14 or 28 days, respectively. Breath and flatus H₂ excretion for 24 h was measured. 1.04 g of resistant fraction in HAS was utilized for 24 h by colonic bacteria. High H₂ excretion was not maintained for 24 h in rats fed the 10% HAS diet, from which only 0.89 g of resistant starch was estimated to be delivered. High colonic H₂ production for 24 h would be maintained by delivering more HAS to the large intestine than is utilized.     

Synergistic roles of leaf boron and calcium during the growing season in affecting sugar and starch accumulation in ripening apple fruit

Fruit sugar content is one of the most important flavor quality traits in the fresh market. Minerals, such as boron (B) and calcium (Ca), are associated with fruit sugar and starch accumulation in many plant species. To better understand the roles of B and Ca in affecting sugar and starch accumulation in apples, 2 g L^?1 Na₂B₄O₇·10H₂O or 10 g L^?1 CaCl₂ was supplied by foliar spray to 20-year-old ‘Fuji’ (Malus domestica Borkh. cv. Fuji) trees at four developmental stages (fruit set, onset of rapid fruit growth, rapid fruit growth and the end of rapid fruit growth), in 2010–2011. The most effective treatment significantly increasing soluble sugar and starch levels in ripening fruit was the foliar application of 2 g L^?1 Na₂B₄O₇·10H₂O during rapid fruit growth, and the robustness of the effects was confirmed for two cultivars, ‘Fuji’ and ‘Orin’, at three orchards in 2011. Foliar applications of B during the onset of rapid fruit growth and rapid fruit growth, as well as the foliar application of Ca at fruit set, significantly increased the soluble sugar content in ripening fruit. In addition, the B application was effective in increasing the fruit starch content, but Ca was not. Both B and Ca treatments significantly increased the leaf concentrations of the other element at least transiently. However, B and Ca effects on fruit sugar/starch did not seem to depend on higher leaf B or Ca levels. In conclusion, B and Ca interact in enhancing fruit sugar and starch contents at the fruit ripening stage.     

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H₂ per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2 mmol of H₂/L/h at 20 g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H₂-based mass and an electricity density of more than 3000 W h/kg of starch.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.