Laboratories

Anna Sikora, PhD, DSc, Prof. of IBB PAS

Laboratory of White Biotechnology

Research Scope

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.

Research

Main Scientific Achievements

  • 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.

Research Description

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

Methodology

  • 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.

Selected Publications

    • 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.

Collaborations

    • 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

Prizes and Awards

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

Publications

WALCZAK P., SIKORA A., DETMAN A., DRĘŻEK K., Wywary gorzelniane jako źródło bakterii wykorzystujących do wzrostu etanol. Rozdział w: Nauki ścisłe i przyrodnicze – przegląd wybranych zagadnień, Wydawnictwo Naukowe Tygiel. Red. J. Jędrzejewska, A. Danielewska; ISBN 978-83-67104-13-5, 2021, s. 50-61
SIKORA A., DETMAN A., WIKTOROWSKA-SOWA E., NOSEK M., SZEWCZYK M., NOWAK SZ., PIOTROWSKI J., Beztlenowy rozkład produktów ubocznych i odpadowych przemysłu cukrowniczego jako źródło biopaliw gazowych. Rozdział w: Nauki ścisłe i przyrodnicze – przegląd wybranych zagadnień, Wydawnictwo Naukowe Tygiel. Red. J. Jędrzejewska, A. Danielewska; ISBN 978-83-67104-13-5, 2021, s. 189-203