Regin Rønn

Impact of protozoan grazing on bacterial community structure in soil microcosms.

ABSTRACT The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.

Fifty thousand years of Arctic vegetation and megafaunal diet

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25–15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

Ancient bacteria show evidence of DNA repair

Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability.

Long-term persistence of bacterial DNA

The persistence of bacterial DNA over geological timespans remains a contentious issue. In direct contrast to in vitro based predictions, bacterial DNA and even culturable cells have been reported from various ancient specimens many million years (Ma) old [1–8]. As both ancient DNA studies and the revival of microorganisms are known to be susceptible to contamination [8–10], it is concerning that these results have not been independently replicated to confirm their authenticity. Furthermore, they show no obvious relationship between sample age, and either bacterial composition or DNA persistence, although bacteria are known to differ markedly in hardiness and resistance to DNA degradation [11]. We present the first study of DNA durability and degradation of a broad variety of bacteria preserved under optimal frozen conditions, using rigorous ancient DNA methods [8–10]. The results demonstrate that nonspore-forming gram-positive (GP) Actinobacteria are by far the most durable, out-surviving endosporeformers such as Bacillaceae and Clostridiaceae. The observed DNA degradation rates are close to theoretical calculations [9], indicating a limit of ca. 400 thousand years (kyr) beyond which PCR amplifications are prevented by the formation of DNA interstrand crosslinks (ICLs). The twelve permafrost samples (0-8.1 Ma) investigated were obtained from northeast Siberia and Beacon Valley, Antarctica. DNA preservation at these sites is exceptional due to constant subzero temperatures, largely neutral pH, and anaerobic conditions. Epifluorescence microscopy revealed ~107cells/gram wetweight in the bacterial size range. The cell counts are in agreement with previous results obtained on permafrost [2,3]. 16S rDNA sequences of 120 bp and 600 bp could be reproducibly amplified from samples up to 400–600 kyr, and show an inverse relationship between PCR amplification efficiency and fragment length that is typical of ancient DNA [8–10,12]. Controls for surface contamination during sampling were negative. Chimeric sequences were excluded from analysis, along with sequences that failed a bootstrap test for independent reproducibility [13]. DNA concentrations and taxonomic diversity were found to decrease with age until 400–600 kyr, at which point the percentage of templates with ICLs reached 100% (Figure 1A–C). Sequences from the older samples appear to be a subset of those from younger material, and all identified bacterial taxa are known soil inhabitants, indicating that permafrost is a nonextremophile environment. There were clear age-related patterns in taxon survival across geographically widespread samples (separated up to 1400 km). Sequences of non-sporeforming GP Actinobacteria, affiliated largely to the genus Arthrobacter (99–100% similarity), consistently persisted for the longest time, followed by GP endospore-forming Bacillaceae and Clostridiaceae and finally gram-negative (GN) bacteria, mostly Proteobacteria (Figure 1D).

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate ∼100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be ∼400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.

Bacteria and protozoa in soil microhabitats as affected by earthworms

Abstract The effects of incorporation of elm leaves (Ulmus glabra) into an agricultural sandy loam soil by earthworms (Lumbricus festivus) on the bacterial and protozoan populations were investigated. Three model systems consisting of soil, soil with leaves, and soil with leaves and earthworms, respectively, were compared. The total, viable, and culturable number of bacteria, the metabolic potentials of bacterial populations, and the number of protozoa and nematodes were determined in soil size fractions. Significant differences between soil fractions were shown by all assays. The highest number of microorganisms was found in microaggregates of 2–53 μm and the lowest in the <0.2μm fraction. A major part of the bacteria in the latter fraction was viable, but non-culturable, while a relatively higher number of culturable bacteria was found in the macroaggregates. The number of colony-forming units and 5-cyano-2,3-ditolyl tetrazolim chloride (CTC)-reducing bacteria explained a major part of the variation in the number of protozoa. High protozoan activity and predation thus coincided with high bacterial activity. In soil with elm leaves, fungal growth is assumed to inhibit bacterial and protozoan activity. In soil with elm leaves and earthworms, earthworm activity led to increased culturability of bacteria, activity of protozoa, number of nematodes, changed metabolic potentials of the bacteria, and decreased differences in metabolic potentials between bacterial populations in the soil fractions. The effects of earthworms can be mediated by mechanical mixing of the soil constituents and incorporation of organic matter into the soil, but as the earthworms have only consumed a minor part of the soil, priming effects are believed partly to explain the increased microbial activity.

Quantitative estimation of flagellate community structure and diversity in soil samples.

Heterotrophic flagellates occur in nearly all soils and, in most cases, many different species are present. Nevertheless, quantitative data on their community structure and diversity are sparse, possibly due to a lack of suitable techniques. Previous studies have tended to focus on either total flagellate numbers and biomass, or the identification and description of flagellate species present. With the increased awareness of the role of biodiversity and of food web interactions, the quantification of species within the community and their response to environmental change is likely to become more important. The present paper describes a modification of the most probable number method that allows such a quantification of individual flagellate morphotypes in soil samples. Observations were also made on the biomass of flagellate morphotypes in soil. 20 to 25 morphotypes of heterotrophic flagellates were detectable per gram of two different arable soils, which were treated experimentally to test the technique. One of the soils was fumigated with chloroform vapour for different lengths of time (0, 0.5, 2 or 24 hours); this led to a reduction in the number of morphotypes, in the Shannon diversity index and in the evenness. The other soil was planted with wheat, and while rhizosphere soils contained the same morphotypes as bulk soil, the abundance of individual morphotypes was significantly different and the Shannon diversity index in rhizosphere soils was significantly higher. Soil influenced by an elevated CO2 level likewise differed significantly in morphotype abundance when compared to soil exposed to ambient levels of CO2. The technique recovered more than 80% of the discernible morphotypes and could also be used to quantify amoebal and ciliate communities in a similar way.

Spatial Distribution and Successional Pattern of Microbial Activity and Micro-Faunal Populations on Decomposing Barley Roots

1. With the current trend in agricultural practice to increase the importance of indigenous and added organic matter in the build-up of soil fertility, a better understanding of the dynamics of decomposition activity around organic resources is required. In this study the changes in microbial activity and populations of protozoa and nematodes were followed in decomposing barley root material buried in soil cores, and in three soil fractions with increasing distance from the root material. 2. Respiratory activity was maximal during the first week and decreased throughout the experiment, and at the last sampling after 392 days no significant effect of the roots on respiration was observed. 3. Following root addition microbial activity (dehydrogenase activity and potential denitrification rate) increased rapidly in the root material and in soil up to 1.8 mm from the roots. Microbial activity peaked after 4 days followed by a peak in protozoan numbers after 2 weeks and a peak in the number of nematodes after 6 weeks. 4. Growth potential of bacteria, as indicated by enzyme synthesis during a potential denitrification assay, and average bacterial cell size was larger on the roots than in the soil suggesting a more active bacterial biomass in this fraction. 5. The root effect was very local, and was limited mainly to the soil fraction adjacent to the roots; soil 1.8-5.4 mm from the roots and soil more than 5.4 mm from the roots was hardly affected by the resource. 6. These results demonstrate distinct successional patterns in the microbial food web with a sequence of population development from micro-organisms to protozoa and nematodes.

Population Dynamics of Active and Total Ciliate Populations in Arable Soil Amended with Wheat

ABSTRACT Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.

Transport and fate of Cryptosporidium parvum oocysts in intermittent sand filters.

The transport potential of Cryptosporidiim parvum (C. parvum) through intermittent. unsaturated, sand filters used for water and wastewater treatment was investigated using a duplicated. 2(3) factorial design experiment performed in bench-scale, sand columns. Sixteen columns (dia = 15 cm, L = 61 cm) were dosed eight times daily for up to 61 days with 65,000 C. parvum oocysts per liter at 15 degrees C. The effects of water quality, media grain size, and hydraulic loading rates were examined. Effluent samples were tested for pH, turbidity, and oocyst content. C. parvum effluent concentrations were determined by staining oocysts on polycarbonate filters and enumerating using epifluorescent microscopy. At completion, the columns were dismantled and sand samples were taken at discrete depths within the columns. These samples were washed in a surfactant solution and the oocysts were enumerated using immunomagnetic separation techniques. The fine-grained sand columns (d50 = 0.31 mm) effectively removed oocysts under the variety of conditions examined with low concentrations of oocysts infrequently detected in the effluent. Coarse-grained media columns (d = 1.40 mm) yielded larger numbers of oocysts which were commonly observed in the effluent regardless of operating conditions. Factorial design analysis indicated that grain size was the variable which most affected the oocyst effluent concentrations in these intermittent filters. Loading rate had a significant effect when coarse-grained media was used and lesser effect with fine-grained media while the effect of feed composition was inconclusive. No correlations between turbidity, pH, and effluent oocyst concentrations were found. Pore-sizc calculations indicated that adequate space for oocyst transport existed in the filters. It was therefore concluded that processes other than physical straining mechanisms are mainly responsible for the removal of C. pavum oocysts from aqueous fluids in intermittent sand filters used under the conditions Studied in this research.