Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum

Climate change and environmental issues compel us to find alternatives to the production of molecules of interest from petrochemistry. This study aims at understanding the production of butyrate, hydrogen, and CO₂ from the oxidation of lactate with acetate in Clostridium tyrobutyricum and thus proposes an alternative carbon source to glucose. This specie is known to produce more butyrate than the other butyrate-producing clostridia species due to a lack of solvent genesis phase. The recent discoveries on flavin-based electron bifurcation and confurcation mechanism as a mode of energy conservation led us to suggest a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. While searching for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum, we identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii. A phylogenetic approach encompassing other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirmed these results. A metabolic scheme on the production of butyrate, hydrogen, and CO₂ from the lactate-acetate co-metabolism in C. tyrobutyricum was constructed and then confirmed with data of steady-state continuous culture. This in silico metabolic carbon flux analysis model showed the coherence of the scheme from the carbon recovery, the cofactor ratio, and the ATP yield. This study improves our understanding of the lactate oxidation metabolic pathways and the role of acetate and intracellular redox balance, and paves the way for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum.

     

Enhanced butanol production in a microbial electrolysis cell by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> IB4

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD⁺ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD⁺, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.     

Electrochemical analysis of Clostridium propionicum and its acrylic acid production in microbial fuel cells

Currently, acrylic acid is produced at a low yield by the resting cells of Clostridium propionicum with the supplement of extra electron acceptors. As an alternative way, acrylic acid production coupled with electricity generation was achieved by C. propionicum‐based microbial fuel cells (MFCs). Electricity was generated in the salt‐bridge MFCs with cysteine and resazurin in the anode chamber as mediators, and K₃Fe(CN)₆ as the cathode electron acceptor. Power generation was 21.78 mW/m² with an internal resistance of 9809 Ω. Cyclic voltammograms indicated the main mechanism of power production was the electron transfer facilitated by mediators in the system. In the salt‐bridge MFC system, 0.694 mM acrylic acid was produced together with electricity generation.     

A genetic and metabolic approach to redirection of biochemical pathways of Clostridium butyricum for enhancing hydrogen production

Clostridium butyricum, a well known H₂ producing bacterium, produces lactate, butyrate, acetate, ethanol, and CO₂ as its main by‐products from glucose. The conversion of pyruvate to lactate, butyrate and ethanol involves oxidation of NADH. It was hypothesized that the NADH could be increased if the formation of these by‐products could be eliminated, resulting in enhancing H₂ yield. Herein, this study aimed to establish a genetic and metabolic approach for enhancing H₂ yield via redirection of metabolic pathways of a C. butyricum strain. The ethanol formation pathway was blocked by disruption of aad (encoding aldehyde‐alcohol dehydrogenase) using a ClosTron plasmid. Although elimination of ethanol formation alone did not increase hydrogen production, the resulting aad‐deficient mutant showed approximately 20% enhanced performance in hydrogen production with the addition of sodium acetate. This work demonstrated the possibility of improving hydrogen yield by eliminating the unfavorable by‐products ethanol and lactate. Biotechnol. Bioeng. 2013; 110: 338–342. © 2012 Wiley Periodicals, Inc.     

Control of the selectivity of butyric acid production and improvement of fermentation performance withClostridium tyrobutyricum

Summary The parameters that control fermentation performance of butyrate production have been studied with a selected strain ofClostridium tyrobutyricum. Fed-batch supply of glucose increased productivity for butyrate. The ratio of butyrate to total acids was strongly influenced by the growth rate of the bacteria, acetate being produced along with butyrate at higher growth rates. In glucose-limited, fed-batch cultures, initially produced acetate was re-utilized, resulting in exclusive production of butyrate. In cultures with non-limiting glucose feeding, the butyrate concentration reached 42.5 g·1^–1 with a selectivity of 0.90, a productivity of 0.82 g·^–1 per hour and a yield of 0.36 g·g^–1 The effects of the mode of supply of glucose on the production of butyrate and acetate are discussed in relation with the energy requirements for cell growth.     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001     

Enhanced electron transfer of different mediators for strictly opposite shifting of metabolism in Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor

Microbial electrosynthesis or electro-fermentation in bioelectrochemical systems (BES) have recently received much attention. Here, we demonstrate with the glycerol metabolism by Clostridium pasteurianum that H ₂ from in situ water electrolysis, especially in combination with a redox mediator, provides a simple and flexible way for shifting product selectivity and enhancing product yield in the fermentation process. In particular, we report and quantify for the first time strictly different effects of Neutral Red (NR) and the barely studied redox mediator Brilliant Blue (BB) on the growth and product formation of C. pasteurianum grown on glycerol in a newly developed BES. We were able to switch the product formation pattern of C. pasteurianum with a concentration-dependent addition of NR and BB under varied iron availability. Interestingly, NR and BB influenced the glycerol metabolism in a strictly opposite manner concerning the formation of the major products 1,3-propanediol (1,3-PDO) and n-butanol (BuOH). Whereas, NR and iron generally enhance the formation of BuOH, BB favors the formation of 1,3-PDO. In BES the metabolic shifts were enhanced, leading to a further increased yield by as high as 33% for BuOH in NR fermentations and 21% for 1,3-PDO in BB fermentations compared with the respective controls. For the first time, the electron transfer mediated by these mediators and their recycle (recharge) were unambiguously quantified by excluding the overlapping effect of iron. BB has a higher capacity than NR and iron. The extra electron transfer by BB can account for as high as 30–75% of the total NAD ⁺ regeneration under certain conditions, contributing significantly to the product formation.     

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E ₀) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E ₀, and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.     

Two differentially regulated nitrate reductases required for nitrate-dependent,microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O₂, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NR_I) and 200 kDa (NR_II). NR_I and NR_II were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO₃. However, only NR_I was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O₂ tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NR_I, while the other two mutants, GRF3 and GRF4, expressed mostly NR_II. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NR_I) or by both microaerobiosis and nitrate (NR_II).Abbreviations NR Nitrate reductase - M _r Relative molecular mass - PMSF Phenylmethylsulfonyl fluoride     

Butyrate production in continuous culture of Clostridium tyrobutyricum: effect of end-product inhibition

Summary Production of butyrate has been studied in continuous cultures of Clostridium tyrobutyricum. Production of acids, gases and cell biomass were determined under conditions of glucose limitation by varying either the glucose input or the dilution rate. Addition of acetate or butyrate to the cultures was also tested. The results led to the proposition that inhibition by acids acting as incouplers of energy production could provide a physiological explanation for most of the phenomena observed. It readily accounted for the higher productivities but lower product concentrations obtained in continuous culture with respect to batch or fed-batch conditions. It also explained the decrease in the ratios of butyrate to total acids and in cell yield observed at higher glucose input as well as the behaviour of the cultures under conditions of excess glucose. It could also possibly account for the partial conversion of added acetate to butyrate observed at moderate growth rates. Offprint requests to: J. P. Vandecasteele     

Inactivation of Sunflower NADH:Nitrate Reductase by White Light-Activated Rose Bengal

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

Mixotrophic chain elongation with syngas and lactate as electron donors

Feeding microbial communities with both organic and inorganic substrates can improve sustainability and feasibility of chain elongation processes. Sustainably produced H₂, CO₂, and CO can be co-fed to microorganisms as a source for acetyl-CoA, while a small amount of an ATP-generating organic substrate helps overcome the kinetic hindrances associated with autotrophic carboxylate production. Here, we operated two semi-continuous bioreactor systems with continuous recirculation of H₂, CO₂, and CO while co-feeding an organic model feedstock (lactate and acetate) to understand how a mixotrophic community is shaped during carboxylate production. Contrary to the assumption that H₂, CO₂, and CO support chain elongation via ethanol production in open cultures, significant correlations (p < 0.01) indicated that relatives of Clostridium luticellarii and Eubacterium aggregans produced carboxylates (acetate to n-caproate) while consuming H₂, CO₂, CO, and lactate themselves. After 100 days, the enriched community was dominated by these two bacteria coexisting in cyclic dynamics shaped by the CO partial pressure. Homoacetogenesis was strongest when the acetate concentration was low (3.2 g L⁻¹), while heterotrophs had the following roles: Pseudoramibacter, Oscillibacter, and Colidextribacter contributed to n-caproate production and Clostridium tyrobutyricum and Acidipropionibacterium spp. grew opportunistically producing n-butyrate and propionate, respectively. The mixotrophic chain elongation community was more efficient in carboxylate production compared with the heterotrophic one and maintained average carbon fixation rates between 0.088 and 1.4 g CO₂ equivalents L⁻¹ days⁻¹. The extra H₂ and CO consumed routed 82% more electrons to carboxylates and 50% more electrons to carboxylates longer than acetate. This study shows for the first time long-term, stable production of short- and medium-chain carboxylates with a mixotrophic community.     

Effect of controlled substrate feeding on butyric acid production byClostridium tyrobutyricum

Summary Production of butyric acid from wheat flour hydrolysate was studied withClostridium tyrobutyricum. The mode of substrate supply was found a key parameter for fermentation performance as large improvements were obtained by feeding with a non-limiting supply of substrate. With this procedure, increases in product concentration and productivity but also in selectivity and yield for butyrate were obtained. Substrate feeding controlled by the rate of gas production was found preferable to constant rate feeding for reason of convenience and flexibility. In these conditions, a butyrate concentration of 62.8 gl^–1 was obtained with a productivity of 1.25 gl^–1 h^–1, a selectivity of 91.5% and a yield of 0.45 g per g of glucose.     

Biosynthesis of butyric acid by Clostridium tyrobutyricum

Butyric acid (C₃H₇COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

Butyric acid production from brown algae using <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> ATCC 25755

Butyric acid fermentation by Clostridium tyrobutyricum ATCC 25755 using glucose or brown algae as a carbon source was carried out. Initially, different fermentation modes (batch, fed-batch, and semi-continuous) at pH 6 and 37°C were compared using a model medium containing glucose as a carbon source. By feeding the whole medium containing 40 ∼ 50 and 30 g/L of glucose into the fed-batch and semi-continuous fermentations, very similar butyrate yields (0.274 and 0.252 g butyrate/g glucose, respectively) and productivities (0.362 and 0.355 g/L/h, respectively) were achieved. The highest butyrate concentration was about 50 g/L, which was observed in the fed-batch fermentation with whole medium feeding. However, semi-continuous fermentation sustained a longer fermentation cycle than the fed-batch fermentation due to end-product and metabolic waste inhibition. The established conditions were then applied to the fermentation using brown algae, Laminaria japonica and Undaria pinnatifida, as substrates for butyric acid fermentation. To hydrolyze brown algae, 7.5 ∼ 10% (w/v) dried brown algae powder was suspended in 1% (w/v) NaOH or 0.5 ∼ 2.5% (w/v) H₂SO₄ and then autoclaved at 121°C for 30 ∼ 90 min. The resulting butyrate concentration was about 11 g/L, which was produced from 100 g/L of L. japonica autoclaved for 60 min in 1.5% H₂SO₄ acid solution.     

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum

Summary When continuous, steady-state, glucose-limited cultures ofClostridium acetobutylicum were sparged with CO, the completely or almost completely acidogenic fermentations became solventogenic. Alcohol (butanol and ethanol) and lactate production at very high specific production rates were initiated and sustained without acetone, and little or no acetate and butyrate formation. In one fermentation, strong butyrate uptake without acetone formation was observed. Growth could be sustained even with 100% inhibition of H₂ formation. Although CO gasing inhibited growth up to 50%, and H₂ formation up to 100%, it enhanced the rate of glucose uptake up to 300%. TheY _ATP was strongly affected and mostly reduced with respect to its steady-state value. The results support the hypothesis that solvent formation is triggered by an altered electron flow.     

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B₁ (AFB₁) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR₅₀ was 20 mM for DMN and 0.072 µM for AFB₁ in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB₁ with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR₅₀ values were 100 mM DMN and 1.8 µM AFB₁ respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.Abbreviations NR Neutral Red - MEM Eagle's Minimum Essential Medium - DMN dimethylnitrosamine - AFB₁ aflatoxin B₁ - LDH lactate dehydrogenase - HBSS Hanks balanced salt solution; - EDTA ethylene bis (oxyethylenenitrilo)-tetraacetic acid - L-15 Leibovitz's 15 - NADH B-nicotinamide adenine dinu - FBS fetal bovine serum - IA immediate autopsy Contribution No. 2816 from Laboratory of Genotoxicology.