Milan Bartoš

Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms

Abstract The production of high-quality methane depends on many factors, including temperature, pH, substrate, composition and relationship of the microorganisms. The qualitative and quantitative composition of methanogenic and sulfate-reducing microorganisms and their relationship in the experimental bioreactors has never been studied. The aim of this research was to characterize, for the first time, the diversity of the methanogenic microorganisms and sulfate-reducing bacteria, and study their relationship and biogas production in experimental bioreactors. Amplification of 16S rRNA gene fragments was carried out. Purified amplicons were paired-end sequenced on an Illumina Mi-Seq platform. The dominant morphotypes of these microorganisms in the bioreactor were homologous (99%) by the sequences of 16S rRNA gene to the Methanosarcina, Thermogymnomonas, Methanoculleus genera and Archaeon deposited in GenBank. Three dominant genera of sulfate-reducing bacteria, Desulfomicrobium, Desulfobulbus and Desulfovibrio, were detected in the bioreactor. The phylogenetic trees showing their genetic relationship were constructed. The diversity and number of the genera, production of methane, hydrogen sulfide and hydrogen in the bioreactor was investigated. This research is important for understanding the relationship between methanogenic microbial populations and other bacterial physiological groups, their substrate competition and, in turn, can be helpful for controlling methanogenesis in bioreactors.

The diversity of sulfate-reducing bacteria in the seven bioreactors

Anaerobic technology has a wide scope of application in different areas such as manufacturing, food industry, and agriculture. Nowadays, it is mainly used to produce electrical and thermal energy from crop processing, solid waste treatment or wastewater treatment. More intensively, trend nowadays is usage of this technology biodegradable and biomass waste processing and biomethane or hydrogen production. In this paper, the diversities of sulfate-reducing bacteria (SRB) under different imputed raw material to the bioreactors were characterized. These diversities at the beginning of sampling and after cultivation were compared. Desulfovibrio, Desulfobulbus, and Desulfomicrobium genus as dominant among sulfate reducers in the bioreactors were detected. The Desulfobulbus species were dominant among other SRB genera before cultivation, but these bacteria were detected only in three out of the seven bioreactors after cultivation dominant.

Detection of mycobacteria in the environment of the Moravian Karst (Bull Rock Cave and the relevant water catchment area): the impact of water sediment, earthworm castings and bat guano

The presence of mycobacteria was studied in Bull Rock Cave (“Byci skala”) and the water catch - ment area of Jedovnice Brook (“Jedovnicky potok”) using direct microscopy after Ziehl-Neelsen (ZN) staining, culture examination and molecular techniques. Mycobacteria were detected in 47.1% of a total of 68 samples. The mycobacterial genes hsp65 and dnaA were detected and sequenced in 37 (74.0%) out of the 50 cave environmental samples and in 10 (55.6%) out of the 18 samples of water catchment sediments. Nine species of slowly growing mycobacteria ( M. terrae , M. arupense , M. gordonae , M. lentiflavum , M. parascrofulaceum , M. parmense , M. sas - katchewanense , M. simiae and M. xenopi ) and two subsp. ( M. avium subsp. avium and M. avium subsp. hominis - suis ) were detected. Fourteen species of rapidly growing mycobacteria ( M. chelonae , M. chubuense , M. poriferae , M. flavescens , M. fortuitum , M. porcinum , M. rhodesiae , M. gilvum , M. goodii , M. peregrinum , M. mageritense , M. vanbaalenii , M. gadium and M. insubricum ) were detected. The highest mycobacterial presence was docu- mented by ZN staining and/or culture examinations in earthworm castings and bat guano (73.3% positivity out of the 15 samples) in the cave environment and in the water sediments collected under the outflow from the wastewater treatment plants (77.8% positivity out of nine samples). The highest total organic carbon (TOC) was detected in wooden material and earthworm castings with pH values between 5.0 and 7.7 in the cave environment and in water sediments collected under the outflow from the wastewater treatment plants with pH between 5.8 and 7.0. It could be concluded that the karst cave environment with its running surface water contaminated with different microorganisms or chemical substances creates favourable conditions not only for animals (especially earthworms) but also for mycobacteria. This fact is also demonstrated by the presence of these mycobacteria in the cave environment mainly in earthworm castings and bat guano.

A new combination of substrates: biogas production and diversity of the methanogenic microorganisms

Abstract Agriculture, food industry, and manufacturing are just some of the areas where anaerobic technology can be used. Currently, anaerobic technologies are mainly used for wastewater treatment, solid waste treatment, or for the production of electrical and thermal energy from energy crops processing. However, a clear trend is towards more intensive use of this technology in biomass and biodegradable waste processing and hydrogen or biomethane production. An enormous number of anaerobic digesters are operating worldwide but there is very little information about the effect of different substrate combinations on the methanogens community. This is due to the fact that each of the anaerobic digesters has its own unique microbial community. For the most effective management of anaerobic processes it would be important to know the composition of a consortium of anaerobic microorganisms present in anaerobic digesters processing different input combinations of raw material. This paper characterizes the effect of the input raw materials on the diversity of the methanogen community. Two predominant microorganisms in anaerobic digesters were found to be 99% identity by the sequences of the 16S rRNA gene to the Methanoculleus and Thermogymnomonas genera deposited in GenBank.

Model-based Characterization of the Parameters of Dissimilatory Sulfate Reduction Under the Effect of Different Initial Density of Desulfovibrio piger Vib-7 Bacterial Cells.

The objective of this study was to design a model of dissimilatory sulfate reduction process using the Verhulst function, with a particular focus on the kinetics of bacterial growth, sulfate and lactate consumption, and accumulation of hydrogen sulfide and acetate. The effect of the initial density (0.12±0.011, 0.25±0.024, 0.5±0.048 and 1.0±0.096 mg cells/ml of medium) of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 on the growth and dissimilatory sulfate reduction was studied. The exponential growth phase of the D. piger Vib-7 was observed for 72 hours of cultivation at the (0.12 and 0.25 mg/ml) initial concentration of bacterial cells. Sulfate and lactate were consumed incompletely during this time. The increase in the initial concentration of cells to 0.5 and 1 mg/ml led to a shortening of the exponential bacterial growth phase and a shift to the stationary phase of the growth. In the case of 0.5 mg/ml seeding, the stationary growth phase was observed in the 36th hour of cultivation. The increase in the initial concentration of cells to 1 mg/ml led to the beginning of the stationary growth phase in 24th hours of cultivation. Under these conditions, sulfate and lactate were consumed completely in the 48th hour of cultivation. The kinetic analysis of the curves of bacterial growth and the process of dissimilatory sulfate reduction by D. piger Vib-7 was carried out.

Nontuberculous mycobacteria in the environment of Hranice Abyss, the world’s deepest flooded cave (Hranice karst, Czech Republic)

Nontuberculous mycobacteria (NTM) are widely distributed in the environment. On one hand, they are opportunistic pathogens for humans and animals, and on the other hand, they are effective in biodegradation of some persistent pollutants. Following the recently recorded large abundance of NTM in extreme geothermal environments, the aim of the study was to ascertain the occurrence of NTM in the extreme environment of the water zone of the Hranice Abyss (HA). The HA mineral water is acidic, with large concentrations of free CO2, and bacterial slimes creating characteristic mucilaginous formations. Both culture and molecular methods were used to compare the mycobacterial diversity across the linked but distinct ecosystems of HA and the adjacent Zbrašov Aragonite Caves (ZAC) with consideration of their pathogenic relevance. Six slowly growing NTM species (M. arupense, M. avium, M. florentinum, M. gordonae, M. intracellulare) and two rapidly growing NTM species (M. mucogenicum, M. sediminis) were identified in the water and in the dry zones at both sites. Proteobacteria were dominant in all the samples from both the HA and the ZAC. The bacterial microbiomes of the HA mineral water and HA slime were similar, but both differed from the microbiome in the ZAC mineral water. Actinobacteria, a phylum containing mycobacteria, was identified in all the samples at low proportional abundance. The majority of the detected NTM species belong among environmental opportunistic pathogens.

Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli

This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.