Selected issues of anaerobic digestion based on the studies on hydrogen- and methane yielding bioreactors

Anaerobic digestion of organic matter results from the metabolic activity of many groups of microorganisms. Interactions between microorganisms during acidogenesis, acetogenesis and methanogenesis, source of inoculum, type of feedstock and operational conditions determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. In innovative installations it is desirable to separate acidogenesis from acetogenesis and methanogenesis to favour respectively the production of biohydrogen or biomethane under controlled conditions

Unsymmetrically Substituted Dibenzo[b,f][1,5]-diazocine-6,12(5H,11H)dione—A Convenient Scaffold for Bioactive Molecule Design

A novel approach for the synthesis of unsymmetrically substituted dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones has been developed. This facile three-step method uses variously substituted 1H-benzo[d][1,3]oxazine-2,4-diones (isatoic anhydrides) and 2-aminobenzoic acids as a starting materials. The obtained products were further transformed into N-alkyl-, N-acetyl- and dithio analogues. Developed procedures allowed the synthesis of unsymmetrical dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones and three novel heterocyclic scaffolds: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)dione, pyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)dione. For 11 of the compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer (HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined. The obtained dibenzo[b,f][1,5]diazocine(5H,11H)6,12-dione framework could serve as a privileged structure for the drug design and development

Synthesis of derivatives of methoxydibenzo[b, f]oxepine in the presence of sodium azide

Dibenzo[b,f]oxepine is an important scaffold in medicinal chemistry and its derivatives occur in several medicinally important plants. A new approach to methoxydibenzo[b,f]oxepines (15–21) proceeding under mild reaction conditions, has been developed. Notably, the use of sodium azide in the reaction allows access to new substituted dibenzo[b,f]oxepines. In order to study their shape and conformation, the optimum structures of these compounds were calculated using the DFT B3LYP/6-311++G(2d,p) method. A docking simulation was performed to insert compound 20 into the crystal structure of tubulin at the colchicine binding site to determine the probable binding model. The information from this work can be helpful for the investigation of new tubulin polymerization inhibitors exhibiting stronger activity

Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart

Searching for Metabolic Pathways of Anaerobic Digestion: A Useful List of the Key Enzymes

The general scheme of anaerobic digestion is well known. It is a complex process promoted by the interaction of many groups of microorganisms and has four major steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The aim of the study was to prepare a systematized list of the selected enzymes responsible for the key pathways of anaerobic digestion based on the Kyoto Encyclopedia of Genes and Genomes database resource. The list contains (i) key groups of hydrolases involved in the process of degradation of organic matter; (ii) the enzymes catalyzing reactions leading to pyruvate formation; (iii) the enzymes of metabolic pathways of further pyruvate transformations; (iv) the enzymes of glycerol transformations; (v) the enzymes involved in transformation of gaseous or nongaseous products of acidic fermentations resulting from nonsyntrophic nutritional interactions between microbes; (vi) the enzymes of amino acid fermentations; (vii) the enzymes involved in acetogenesis; and (viii) the enzymes of the recognized pathways of methanogenesis. Searching for the presence and activity of the enzymes as well as linking structure and function of microbial communities allows to develop a fundamental understanding of the processes, leading to methane production. In this contribution, the present study is believed to be a piece to the enzymatic road map of anaerobic digestion research

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNArepair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes.Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. Theaim of this study was to assess the usefulness of these alkB mutants in different aspects of research onAlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes incells. Using of HK82 and BS87 strains, we found the following differences among these alkB−derivatives:(i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii)different specificity of Arg+revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv)the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, andnuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, mayresult in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis.In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearingseveral additional mutations in its genome, is not an exact derivative of the AB1157 strain, and showsadditional features that may disturb proper interpretation of obtained results

Evaluation of Anti-cancer Activity of Stilbene and Methoxydibenzo[b,f] oxepin Derivatives

Background: Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. Objective: We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo[b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. Results: Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair, and stress response, e. g. oxidative stress. Conclusions: Among newly synthesized stilbene derivatives we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis, and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives

Inhibition of hydrogen-yielding dark fermentation by ascomycetous yeasts

Hydrogen-yielding fermentation conducted in bioreactors is an alternative method of hydrogen production. However, unfavourable processes can seriously inhibit bio-hydrogen generation during the acidogenic step of anaerobic digestion. Here, ascomycetous yeasts were identified as a major factor inhibiting the production of bio-hydrogen by fermentation.Changes in the performance of hydrogen-producing bioreactors including metabolic shift,quantitative changes in the fermentation products, decreased pH, instability of the microbialcommunity and consequently a dramatic drop in bio-hydrogen yield were observed following yeast infection. Ascomycetous yeasts from the genera Candida, Kazachstania and Geotrichum were isolated from hydrogen-producing bioreactors. Yeast metabolites secreted into the growth medium showed antibacterial activity. Our studies indicate that yeast infection of hydrogen-producing microbial communities is one of the serious obstacles to use dark fermentation as an alternative method of bio-hydrogen production. It also explains why studies on hydrogen fermentation are still limited to the laboratory or pilot-scale systems

Lignite biodegradation under conditions of acidic molasses fermentation

Lignite is difficult to degrade, thus stimulation of the autochthonous lignite microflora and introduction of additional microorganisms are required for lignite decomposition. Here, a packed bed reactor, filled with lignite samples from the Konin region (central Poland) was supplied continuously with M9 medium, supplemented with molasses (a by-product from the sugar industry), for 124 days to stimulate the autochthonous lignite microflora. Acidic fermentation of molasses was observed in the bioreactor. The simultaneous decomposition of lignite occurred under this acidic molasses fermentation condition. Our results show decay of free (non-bound) organic compounds during anaerobic lignite biodegradation. The concentrations of n-alkanes, n-alkanols, n-alkanoic acids, diterpenoids, triterpenoids and steroids present in non-biodegraded samples decreased significantly (some compounds to zero) during biodegradation. Interestingly, other compound classes like phenols, ketones and certain organic compounds increased. We interpret this phenomenon as a gradual decomposition of polymers, lignin and cellulose, present in the lignite. These changes resulted from microbial activity since they were not observed in pure solutions of short-chain fatty acids. The 16SrRNA profiling of the microbial community selected in the bioreactor revealed that the dominant bacteria belonged to the Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes, furthermore representatives of 16 other phyla were also found. All the known taxa of lignocellulolytic bacteria were represented in the microbial community. Synergistic relations between bacteria fermenting molasses and bacteria degrading lignite are assumed. The results confirm lignin degradation in acidic medium by bacteria under anaerobic conditions