This investigation has for its aim the confirmation of the correlation between soil moisture and the microflora of soils, and a lesser relation between the soil temperature and the microflora. The soils used in this study are typical clay soils of Denton County, Texas. The samples were taken from virgin pasture lands and analyses made monthly or semimonthly. The wide seasonal variation of temperature and rainfall afford a good opportunity for this study.

Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.

MSc (Botany) / Department of Botany

11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

The soil microbial community has a major impact on ecosystem processes on a global scale. Anthropogenic stress has a significant effect on the composition, biomass and activity of the microbial community. In addition, this effect depends not only on the anthropogenic activity, but also on the environmental conditions. Modern analytical and molecular methods, including the use of biochemical markers, can be well used to monitor changes in the microbial community. These methods do not monitor the community directly, but detect the substances that are secreted and transformed by microbes and, last but not least, those that are part of them. The amount of these biochemical markers reflects the biomass, condition and taxonomic composition of a particular microbial community. In the presented work, these markers were used to monitor the response of microorganisms in various ecosystems which are affected directly or indirectly by human activity. The results of the dissertation are presented in four articles. Three of them have been published in international journals with IF and one is prepared in the form of a manuscript for publication. The first publication presents the results of research exploring the impact of traditional agriculture in Papua New Guinea on the soil microbial communities, soil...

Sulfonamides are antibiotics that are frequently used both in human and veterinary medicine. The combinations of abundant use of these antibiotics and their natural resistance to decay leads often to long term persistence in the environment. This accumulation, especially in living organisms, may lead to subsequent toxicosis. Also, presence of these antibiotic in nature poses problems with regard to the spread of genes for antibiotic resistance between potential pathogens. These facts led to an increase interest in studying the sulfonamide biodegradation and subsequent sulfonamide removal from the environment. In this work it was possible to isolate and characterize the microorganism Acinetobacter sp. strain 49. This microorganism was able to biodegrade under right conditions sulfamethoxazole with 80 % efficiency. Sulfamethoxazole is one of the most commonly found sulfonamide in the nature.

Cellulase-producing microorganisms are considered as a key player in various environments to degrade the plant biomass and were isolated from various environment like soils, mangroves and oceans. The Red Sea has a unique environment in terms of high seawater temperature, high salinity and low nutrients. This study aims of examining if the Red Sea is a potential resource for cellulase-producing microorganisms and cellulase genes. First, I investigated types of microbial cellulase genes in the Red Sea based on public metagenomic datasets. The analysis revealed 3,383 microbial cellulase were more abundant in shallow depth than in deep seawater, and were classified into 16 sub-GH orthologous groups. These results suggest that the Red Sea environment is potentially an excellent gene resource of microbial cellulases due to its high diversity. Next, cellulase-producing microorganisms were isolated and screened from the Red Sea. Three bacterial and one fungal strain were successfully obtained. The MLTS analysis showed that the three bacterial strains belong to Bacillus paralichiniformis. The 18S rRNA of fungal strain showed 99% similarity to Aspergillus ustus and the enzymatic assay of the four strains showed high cellulase activity. These results suggest that these four isolates secreted active cellulases. Next, I tried to identify cellulase genes actually working during their cellulolysis by conducting comparative transcriptome analysis of the candidate genes and identified cellulase genes that are highly expressed during cellulolysis. To my knowledge, it is the first attempt to find out cellulase genes functioning during their cellulolysis among distinct cellulases on genomes of microorganisms. The results showed that although all the candidate genes were upregulated in general, a limited number of cellulase genes were highly expressed, which are highly expected to have a crucial role in cellulolysis. I also identified operon structures composed of genes including cellulases. This will provide us with the information to elucidate the cellular mechanisms occurring along with the cellulolysis in bacterial strains. We can expect that the Red Sea is a potential resource for new cellulase genes applicable for the industry. These information can be significantly useful for the bio-prospecting research of microbial cellulases in the Red Sea.

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

This study investigated the use of electroporation as an extraction method of lipids in oleaginous microorganisms. Electroporation is the process of placing a voltage gradient across a lipid membrane to create pores that vary in size and longevity with voltage magnitude and pulse duration. Once the voltage gradient is removed, the lipid membrane will seal the pore. The use of electroporation on oleaginous microorganisms to extract stored lipids could be a useful tool if the microorganism is allowed to regenerate and produce more lipids. Three high-lipid media were investigated: soybeans, Rhodococcus opacus (bacteria), and Rhodotorula glutinis (yeast). This study investigates varied voltage magnitude, pulse duration, quantity of pulses, and distance between electrodes. Electroporation proved to be moderately successful for lipid removal when using low voltages and long pulse durations. However, electroporation removed only a small percentage of the intracellular lipids.

Microbial contaminants in water are a major public health concern. Pathogens have been identified as a primary threat to river water quality in the United States, potentially impacting drinking and irrigation water sources and recreational waters. Agricultural runoff, feedlot operations, wastewater effluents, swimming activities, domestic and wild animals are potential sources of microbial contamination. This thesis presents Massachusetts as a case study for linking public health data of waterborne gastrointestinal diseases with sources of drinking water, potential recreational exposures, as well as hydrologic, climatic, and land use data. Giardia sp. has been chosen as a model organism. Information of reported human Giardiasis cases has been synthesized. Using Geological Information system and statistical software (SPSS and SAS) relationships of confirmed Giardiasis have been compared with available climate and hydrologic data. In this thesis the research finding suggest that there is no visible difference in disease occurrence related with amount of precipitation or extreme rain event. However human giardiasis in Massachusetts has been found related with temperature thus shows a seasonal trend in disease occurrence. Seasonal water related human activity likely have played a role in disease occurrence.