Pracownie badawcze

Dr hab. Anna Sikora, Prof. IBB PAN

Pracownia Białej Biotechnologii

ORCID: 0000-0002-9464-6851

Zakres badań

Our laboratory is interested in microbial interactions and functions in built-environment microbiomes, especially anaerobic digestion microbiomes. The long-term goal of our studies is the microbial production of biohydrogen and biomethane for energy and the scale-up of these systems from the bench to industrial installations. We seek to recognize stages of anaerobic digestion at the molecular level and develop knowledge-based bio-economy.

Badania

Najważniejsze osiągnięcia badawcze

  • We elaborated an innovative two-stage system for microbial H2 and CH4 production based on the anaerobic digestion of byproducts of the sugar industry.
  • Using C. butyricum as a new model, we proposed an updated metabolic scheme of lactate and acetate conversion to butyrate, which employs flavin-based electron bifurcation.
  • We identified a community balance between HPB and LAB as a crucial factor that determines stable H2-producing systems.
  • We described ascomycetous yeast as a new factor that inhibits microbiological H2 production in DF bioreactors under mesophilic conditions.
  • We proposed a new model for studies of acetogenic and methanogenic steps of AD.

Opis badań

The anaerobic digestion (AD) of biomass to CH4 and CO2 is used for the production of renewable energy. Dark fermentation (DF), which forms part of the acidogenic step of AD, is a biological method of bio-H2 production. Modern biogas plants are being constructed, in which the H2-yielding (acidogenesis) and CH4-yielding (acetogenesis and methanogenesis) stages of AD are separated to generate these gases under controlled conditions.

Research on two-stage AD in our laboratory has been conducted since 2010. We developed a bench-scale two-stage AD system that produces bio-H2 and biogas (bio-CH4) from sucrose-rich byproducts of the sugar beet industry under mesophilic conditions. The system has been successfully scaled up 10-fold and currently operates in the Dobrzelin sugar factory (Polish Sugar Company KSC S.A.) as a research facility for a 200-fold-enlarged installation (ongoing project). The aims of the ongoing project are the estimation of economic effectiveness of bio-H2 and biogas production from byproducts of the sugar industry, evaluation of the method of bio-H2 storage in metal hydrides, the presentation of different applications of the produced fermentation gases, and an increase in the contribution of renewable energy to the overall energy pool. Future plans include further collaborations with state-owned companies in the field of the generation of green energy and achievement of a zero-emission economy.

The bench-scale studies were also a source of ideas for basic research on acidogenesis, acetogenesis, and methanogenesis. Interactions between microorganisms during specific steps of AD determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. Several well-recognized unfavorable processes can seriously inhibit bio-H2 generation during acidogenesis. In our studies, ascomycetous yeasts were identified as a new factor that inhibits the production of bio- H2 through a metabolic shift to ethanol and lactic acid fermentation and the secretion of antimicrobial metabolites, such as 1,3-b-glucosidase. These findings open up a new area of research on interactions between bacteria and yeasts, especially in mixed culture fermentations. This subject is relevant to the food technology, human health (probiotics), and antibacterial therapies.

We showed that high-efficiency bio-H2-producing DF microbial communities (MCs) are composed of well-recognized H2-producing bacteria (HPB), such as Clostridiales, Bacillus, Prevotella, and Enterobacteriaceae, and lactic acid bacteria (LAB). We tried to explain the relevance of the LAB in DF MCs. We confirmed that DF MCs that were fed molasses were able to transform lactate and acetate to butyrate, H2, and CO2 in batch experiments and continuous systems. These findings indicate that a phenomenon that is analogous to the cross-feeding of lactate in the gastrointestinal tract also occurs in DF reactors. We proposed a scheme of the lactate and acetate transformation pathway, based on the new model Clotridium butyricum, that employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein complexes that are specific to lactate oxidation and butyrate formation.

Our plans include studies of the conversion of lactate and acetate to butyrate in piglets with non-pathogenic intestinal inflammation and the elaboration of an easy test that is based on this process to diagnose gut inflammation.

Acetogenesis and methanogenesis are highly interconnected processes because of syntrophy between acetogenic bacteria and hydrogenotrophic methanogens. The aims of our research are to determine the influence of dominant products of acidogenesis on metabolic pathways of CH4 formation.

We plan to expand our studies on the selection of microorganisms that are capable of polyethylene and polypropylene degradation to develop new technologies for the controlled decomposition of plastics.

  • Bibliography
  1. Detman et al. Front Microbiol. 2020. DOI: 10.3389/fmicb.2020.612344.
  2. Detman et al. Microbiome. 2020. DOI:10.21203/rs.3.rs-67649/v1. Under review.
  3. Detman et al. Pol. J. Environ. 2017. DOI: https://doi.org/10.15244/pjoes/68149.
  4. Elbeshbishy et al. Renew Sust Energ Rev. 2017. DOI: 10.1016/j.rser.2017.05.075.
  5. Sikora et al. Book Chapter in: Fermentation Processes, book Chapter. 2017. DOI: 10.5772/64645.
  6. Sikora et al. Book Chapter in: Anaerobic Digestion. 2019. DOI: 10.5772/intechopen.81256.
  7. Sikora et al. Book Chapter in: Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes. 2013. DOI: 10.5772/50364.
  8. Stams & Plugge. Nat Rev Microbiol. 2009. DOI: 10.1038/nrmicro2166.
  9. Microbiol. 1998. DOI: 10.1099/00221287-144-9-2377
  10. Weghoff et al. Environ Microbiol. 2015. DOI: 10.1111/1462-2920.12493

Metodologia

  • Microbiological methods

– Anaerobic cultivation of microorganisms: (i) continuous cultivation of hydrogen- and methane-producing MCs in packed bed reactors (PBRs) and up-flow anaerobic sludge blanket (UASB) bioreactors connected to peristaltic pumps, (ii) static batch cultures of microorganisms in a vinyl anaerobic chamber (Coy Laboratory Products, Inc.) or in Labit bioreactors that are equipped with the OxiTop system.
– Selection of microorganisms as MCs or pure cultures with desired properties.
– Microscopic observation using a light microscope (Nikon Eclipse E200; 100× objective lens) equipped with a digital camera.

  • Analytical methods

– Determination of chemical oxygen demand (COD), sludge activity (TTC-dehydrogenase activity of activated-sludge), concentration of sulfide (S2–), soluble iron (Fe2+/3+), total nitrogen, short-chain fatty acids (SCFAs), and total organic carbon using the Compact photometer PF 12 Macherey-Nagel and thermostat for Nanocolor tests.
– Determination of total rate of fermentation gas production using MilliGascounter MGC-1 (RITTER) and Bubble flowmeter ZAM Kęty.
– Analyses of the composition of fermentation gases using an HPR20 Hiden Analytical mass spectrometer (continuous analysis) and Agilent 7820B gas chromatograph (analysis of collected gas samples).
– Analyses of SCFAs and ethanol using an Agilent 7820A gas chromatograph.

  • Molecular biology methods

– DNA and RNA isolation from MCs and environmental samples for further metagenomics and metatranscriptomic analyses.
– Identification of bacteria and yeasts based on the 16S rRNA gene and ITS1 plus 28S rRNA gene sequences, respectively (thermocyclers, NanoDrop 1000 spectrophotometer).

  • Methods in collaboration with indoor service laboratories and other research institutions

DNA sequencing, metagenomic analyses, mass spectrometry analysis of proteins, analyses of stable carbon isotope composition of fermentation gases, analyses of SCFAs and carbohydrates using high-performance liquid chromatography, and scanning electron analysis.

Wybrane publikacje

  • Detman A, Mielecki D, Chojnacka A, Salamon A, Blaszczyk MK, Sikora A. Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors. Microb Cell Fact. 2019. doi: 10.1186/s12934-019-1085-1.
  • Detman A, Mielecki D, Pleśniak Ł, Bucha M, Janiga M, Matyasik I, Chojnacka A, Jędrysek M-O, Błaszczyk MK, Sikora A. Methane‑yielding microbial communities processing lactate‑rich substrates: a piece of the anaerobic digestion puzzle. Biotechnol Biofuels. 2018. doi: 10.1186/s13068-018-1106-z.
  • Detman A, Chojnacka A, Mielecki D, Błaszczyk MK, Sikora A Inhibition of hydrogen-yielding dark fermentation by ascomycetous yeasts. Int J Hydrogen Energy. 2018. doi: 10.1016/j.ijhydene.2018.05.004.
  • Noteworthy facts about a methane-producing microbial community processing acidic effluent from sugar beet molasses fermentation. Chojnacka A, Szczęsny P, Błaszczyk MK, Zielenkiewicz U, Detman A, Salamon A, Sikora A. PLoS One. 2015. doi:10.1371/journal.pone.0128008.
  • Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria. Chojnacka A, Błaszczyk MK, Szczęsny P, Nowak K, Sumińska M, Tomczyk-Żak K, Zielenkiewicz U, Sikora A. Bioresour Technol. 2011. doi:10.1016/j.biortech.2011.08.063.

Współpraca

  • Daniel Laubitz, Arizona Steele Children's Research Center, University of Arizona Health Sciences, Tucson, Arizona, USA
  • Stefano Campanaro, Laura Treu, Department of Biology, University of Padova, Padova, Italy
  • Jan Piotrowski, Polish Sugar Company S. A., Warsaw, Poland
  • Michał Bucha, Leszek Marynowski, Ewa Łupikasza, Faculty of Earth Sciences, University of Silesia in Katowice, Sosnowiec, Poland
  • Wojciech Tokarz, Sieć Badawcza Łukasiewicz – Industrial Chemistry Research Institute, Warsaw, Poland
  • Izabela Witońska, Institute of General and Ecological Chemistry, Faculty of Chemistry, Lodz University of Technology, Lodz, Poland
  • Piotr Dziugan, Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Lodz, Poland
  • Zbigniew Łodziana, Institute of Nuclear Physics Polish Academy of Sciences, Cracow, Poland
  • Agnieszka Salamon, Fruit and Vegetable Products Technology Department, Prof. Wacław Dąbrowski Institute of Agriculture and Food Biotechnology, Warsaw Poland
  • Mirosław Słowakiewicz, Faculty of Geology, University of Warsaw, Warsaw Poland
  • Paweł Flianowski, Czesław Rybicki, Department of Natural Gas Engineering, Faculty of Drilling, Oil & Gas
  • AGH – University of Science and Technology, Cracow, Poland

Nagrody i wyróżnienia

  • Anna Detman. START Fellowship. 2021. Foundation for Polish Science, Poland.
  • Aleksandra Chojnacka, START Fellowship. 2012. Foundation for Polish Science, Poland.

Publikacje (z afiliacją IBB PAN)

SIKORA A., DETMAN A., CHOJNACKA A., BŁASZCZYK M.K., Anaerobic digestion: I. A common process ensuring energy flow and the circulation of matter in ecosystems. II. A tool for the production of gaseous biofuels. Chapter 14 in: Fermentation Processes. Ed. Faustino Jozala A., InTech; ISBN 978-953-51-2927-1 [310p.] 2017, p.271-301 DOI: 10.5772/64645 IF - (2017)
PIELA P., TOKARZ W., KAŹMIERCZAK W., DETMAN A., SIKORA A., PIOTROWSKI J., The use of hydrogen-rich gas obtained from dark fermentation of molasses from sugar industry for fueling a fuel cell. Przemysł Chemiczny (2016) 95(5): 993-999 DOI: 10.15199/62.2016.5.20 IF 0.367
MIELECKI D., SIKORA A., WRZESIŃSKI M., NIEMINUSZCZY J., DETMAN A., ŻUCHNIEWICZ K., GROMADKA R., GRZESIUK E., Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies. DNA Repair (2016) 39: 34-40 DOI: 10.1016/j.dnarep.2015.12.010 IF 3.929
SIKORA A., MACIEJEWSKA A.M., POZNAŃSKI J., PILŻYS T., MARCINKOWSKI M., DYLEWSKA M., PIWOWARSKI J., JAKUBCZAK W., PAWLAK K., GRZESIUK E., Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis (2015) 778: 52-60 DOI: 10.1016/j.mrfmmm.2015.05.009 IF 3.680
CHOJNACKA A., SZCZĘSNY P., BŁASZCZYK M.K., ZIELENKIEWICZ U., DETMAN A., SALAMON A., SIKORA A., Noteworthy facts about a methane-producing microbial community processing acidic effluent from sugar beet molasses fermentation. PLOS ONE (2015) 10(5): e0128008 (23p.) DOI: 10.1371/journal.pone.0128008 IF 3.234
MIELECKI D., SAUMAA S., WRZESIŃSKI M., MACIEJEWSKA A.M., ŻUCHNIEWICZ K., SIKORA A., PIWOWARSKI J., NIEMINUSZCZY J., KIVISAAR M., GRZESIUK E., Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA – In Vivo, In Vitro, and In Silico studies. PLOS ONE (2013) 8(10): e76198 (24 p.) IF 3.730
SIKORA A., BŁASZCZYK M.K., JURKOWSKI M., ZIELENKIEWICZ U., Lactic acid bacteria in hydrogen-producing consortia: on purpose or by coincidence? Chapter 21 in: Lactic Acid Bacteria - R & D for Food, Health and Livestock Purposes. (p.658) Book edited by Marcelino Kongo, ISBN 978-953-51-0955-6; Published: January 2013; p.488-514
SIKORA A., JANION C., GRZESIUK E., Bacterial systems for testing spontaneous and induced mutations. Chapter 8 in: Mutagenesis (p.246) Ed. Rajnikant Mishra, InTech 2012, ISBN 978-953-51-0707-1, p.153-178
CHOJNACKA A., BŁASZCZYK M.K., SZCZĘSNY P., NOWAK K., SUMIŃSKA M., TOMCZYK-ŻAK K., ZIELENKIEWICZ U., SIKORA A., Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria. Bioresource Technology (2011) 102: 10057-10064 IF 4.365
SIKORA A., WÓJTOWICZ-SIEŃKO J., PIELA P., ZIELENKIEWICZ U., TOMCZYK-ŻAK K., CHOJNACKA A., SIKORA R., KOWALCZYK P., GRZESIUK E., BŁASZCZYK M.K., Selection of bacteria capable of dissimilatory reduction of Fe(III) from a long-term continuous culture on molasses and their use in a microbial fuel cell. Journal of Microbiology and Biotechnology (2011) 21(3): 305-316 IF 1.224
SIKORA A., GRZESIUK E., Reversion of argE3 to Arg+ in Escherichia coli AB1157 - an informative bacterial system for mutation detection. Acta Biochimica Polonica (2010) 57(4): 479-485 IF 1,262
PIELA P., MICHAŁOWSKI T., MILTKO R., SZEWCZYK K., SIKORA R., GRZESIUK E., SIKORA A., Can a fermentation gas mainly produced by rumen Isotrichidae ciliates be a potential source of biohydrogen and a fuel for a chemical fuel cell? Journal of Microbiology and Biotechnology (2010) 20(7): 1092-1100 IF 2,062
WRZESIŃSKI M., NIEMINUSZCZY J., SIKORA A., MIELECKI D., CHOJNACKA A., KOZŁOWSKI M., KRWAWICZ J., GRZESIUK E., Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E.coli strains deficient in functional AlkB protein. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis (2010) 688: 19-27 IF 3,556
SIKORA A., MIELECKI D., CHOJNACKA A., NIEMINUSZCZY J., WRZESIŃSKI M., GRZESIUK E., Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair. Mutagenesis (2010) 25(2): 139-147 IF 3,541
NIEMINUSZCZY J., MIELECKI D., SIKORA A., WRZESIŃSKI M., CHOJNACKA A., KRWAWICZ J., JANION C., GRZESIUK E., Mutagenic potency of MMS-induced 1meA/3meC lesions in E.coli. Environmental and Molecular Mutagenesis (2009) 50: 791-799 IF 2,181
SIKORA A., GRZESIUK E., Heat shock response in gastrointestinal tract. Journal of Physiology and Pharmacology (2007) 58 Suppl. 3: 43-62 IF 2,974
LAUBITZ D., JANKOWSKA A., SIKORA A., WOLIŃSKI J., ZABIELSKI R., GRZESIUK E., Gut myoelectrical activity induces heat shock response in Escherichia coli and caco-2 cells. Experimental Physiology (2006) 91: 867-875 IF 2,339
NIEMINUSZCZY J., SIKORA A., WRZESIŃSKI M., JANION C., GRZESIUK E., AlkB dioxygenase in preventing MMS-induced mutagenesis in Escherichia coli: effect of Pol V and AlkA proteins. DNA Repair (2006) 5: 181-188 IF 5,868

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