medium-preparation-for-cultivation-microorganisms-under-strictly

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Universität Innsbruck

A thermographimetric method which allows for a quick and accurate estimation of intra- and extracellular water of microbial cells is reviewed and improved. Knowledge of these fractions is important for physiological as well as for toxicological investigations. Results of the study indicate that besides the species, nutrient availability and growth conditions affect the intracellular water content. Intra- and extracellular water, dry matter, volume and density of a single cell of Arthrobacter sp. are calculated. There are indications that intracellular compartments of eukaryotes could also be investigated with this method.

A practicable and accurate method to differentiate between intra- and extracellular water of microbial cells.

A distinct inhibition of the growth of a typical soil-born bacterium, Arthrobacter sp. PI/1-95, by aluminium was established. The content of intracellular water and (consequently) the cell size of the bacterium significantly increased when cells came into contact with aluminium. Both parameters were also increased when applying HCl, but Al caused the greater change, so that a distinct discrimination between the effects of aluminium on the one hand and a sole pH-effect on the other hand was possible. The presented results point to an osmo-regulative disorder connected with a malfunction of cell membrane and cell wall, which seems to be a probable mechanism for an Al-toxicity in connection with Arthrobacter sp. PI/1-95.

Influence of Al on growth, cell size and content of intracellular water of Arthrobacter sp. PI/1-95.

Two coryneform bacteria Arthrobacter sp. PI/1-95 (Arth1) and Arthrobacter sp. PI/3-95 (Arth3) were isolated from forest soil characterized and investigated with respect to their reaction towards aluminium (Al). Sigmoid functions were used to describe dose-response relationships between Al concentration and microbial growth and to calculate EC-values. EC(100) -- indicating a complete inhibition of microbial growth -- varied between 185 microM Al for Arth1 and 11 mM Al for Arth3. A pure pH effect seems probable in connection with the sensitive Arth1 but not with the tolerant species Arth3. Temperature was shown to distinctly increase the toxic effects of Al towards Arth3 whereas only a moderate modification in Al-toxicity was observed with Arth1.

Effect of temperature and pH on the toxicity of aluminium towards two new, soil born species of Arthrobacter sp.

The influence of iron, aluminium and of the combined application of both metals on microbial biomass and production of siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and Hymenoscyphus ericae) were investigated. All three species showed a strong iron regulation and Al-sensitivity of siderophore biosynthesis although several differences remained species dependent. Inhibitory effects of Fe and Al on siderophore-production were additive and the higher binding capacity of siderophores towards iron could be compensated by a higher Al-availability. Although pH itself is also important for regulation of siderophore biosynthesis, an indirect effect of Al on siderophore production via an Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a reduced biomass production were compensated by high Fe-availability, whereas the addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.

Interactions between iron availability, aluminium toxicity and fungal siderophores.

A 750,000l digester located in Roppen/Austria was studied over a 2-year period. The concentrations and amounts of CH4, H2, CO2 and H2S and several other process parameters like temperature, retention time, dry weight and input of substrate were registered continuously. On a weekly scale the pH and the concentrations of NH4+ -N and volatile fatty acids (acetic, butyric, iso-butyric, propionic, valeric and iso-valeric acid) were measured. The data show a similar pattern of seasonal gas production over 2 years of monitoring. The consumption of VFA and not the hydrogenotrophic CH4 production appeared to be the limiting factor for the investigated digestion process. Whereas the changes in pH and the concentrations of most VFA did not correspond with changes in biogas production, the ratio of acetic to propionic acid and the concentration of H2 appeared to be useful indicators for reactor performance. However, the most influential factors for the anaerobic digestion process were the amount and the quality of input material, which distinctly changed throughout the year.

Effect of seasonal changes in quantities of biowaste on full scale anaerobic digester performance.

A 750,000litre fermenter was studied throughout one entire year by investigating the concentrations of volatile fatty acids (acetic, butyric, i-butyric, propionic, valeric and i-valeric acids), pH, concentrations of total C, N, S and NH(4)(+)-N, amounts of chemical and biological oxygen demand, and abundance of acetogenic microorganisms. Additionally several process parameters such as temperature, retention time, dry weight and input of substrate and liquids, and the concentrations and amounts of CH(4), H(2), CO(2) and H(2)S within the biogas were monitored continuously. Various volatile fatty acids and the ratio of acetic to propionic acid were shown to allow a rough indication on the fermentation but were not sufficiently precise to describe the fermenter performance. Nutrient compounds and special fractions, such as easily extractable carbohydrates or the concentration of total fats were more strongly correlated to the gas production of the fermenter. Results of an MPN-method for the determination of acetogenic microorganisms point to an important role of these microorganisms during the phase of restoration of the fermenter performance.

Process parameters within a 750,000 litre anaerobic digester during a year of disturbed fermenter performance.

PCR-DGGE, qPCR and sequencing highlighted a quite homogenous archaeal community prevailing in secondary calcite deposits, so-called moonmilk, within the cold alpine Hundalm cave in Tyrol (Austria). Furthermore, the depth profile of this moonmilk could prove that the Archaea are located in oxygen-rich near- and oxygen-depleted sub-surface layers. To gather these communities we therefore applied an aerobic and anaerobic cultivation approach in oligotrophic and methanotrophic media. The mixed moonmilk community was analyzed with a combination of molecular methods using qPCR, PCR-DGGE and sequencing. Anaerobic and aerobic cultures were additionally investigated with GC and HPLC analyses. It was possible to initially cultivate and enrich the supposed aerobic/microaerophilic and anaerobic archaeal fraction, representing the natural archaeal community. While the naturally less abundant near-surface Archaea are closely related to members of the Thaumarchaeota (Nitrosopumilus maritimus), the highly abundant anaerobic Archaea are more distantly related to members within the Euryarchaeota. It is possible that these cultivable moonmilk-born Archaea represent new ecotypes or are so far undescribed. Based on the sequencing results and the production of very low amounts of methane, a corresponding methanogenic community is thought to represent only a minor abundant archaeal fraction. On a physiological level the cultivated moonmilk community is cold-adapted and basically of oligotrophic and organotrophic character. 

Cultivation of moonmilk-born non-extremophilic Thaum and Euryarchaeota in mixed culture.

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of  to use carbon dioxide (CO) for catabolic and anabolic metabolism was not proven until now. Here, we show that  used CO to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO consecutively, resulting in a biphasic methane production. Growth exclusively from CO occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from CO into CH molecules was measured to validate the obtained data. New insights into the physiology of  can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Hydrogenotrophic Methanogenesis and Autotrophic Growth of .

Substrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions.

Formation of phenylacetic acid and phenylpropionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

Biological Pretreatment Strategies for Second-Generation Lignocellulosic Resources to Enhance Biogas Production.

Serving as "natural laboratories", altitudinal gradients can be used to study changes in the distribution of microorganisms in response to changing environmental conditions that typically occur over short geographical distances. Besides, rhizosphere zones of plants are known to be hot-spots for microbial diversity and to contain different microbial communities when compared with surrounding bulk soil. To discriminate the effects of altitude and plants, we investigated the microbial communities in the rhizosphere of  and bulk soil along a high-alpine altitudinal gradient (2,600-3,400 m a.s.l.). The research area of this study was Mount (Mt.) "Schrankogel" in the Central Alps of Tyrol (Austria). Our results point to significantly different microbial diversities and community compositions in the different altitudinal belts. In the case of prokaryotes, environmental parameters could explain 41% of the total variation of soil communities, with pH and temperature being the strongest influencing factors. Comparing the effects derived from fraction (bulk vs. rhizosphere soil) and environmental factors, the effects of the roots of  accounted for about one third of the explained variation. Fungal communities on the other hand were nearly exclusively influenced by environmental parameters accounting for 37.4% of the total variation. Both, for altitudinal zones as well as for bulk and rhizosphere fractions a couple of very specific biomarker taxa could be identified. Generally, the patterns of abundance of several taxa did not follow a steady increased or decreased trend along the altitudinal gradient but in many cases a maximal or minimal occurrence was established at mid-altitudes (3,000-3,100 m). This mid-altitudinal zone is a transition zone (the so-called alpine-nival ecotone) between the (lower) alpine grassland/tundra zone and the (upper) sparsely vegetated nival zone and was shown to correspond with the summer snow line. Climate change and the associated increase in temperature will shift this transition zone and thus, might also shift the described microbial patterns and biomarkers.

Paul Illmer

Department of Microbiology

Universität Innsbruck

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions