Spatial and temporal fluctuations in bacteria, microfauna and mineral nitrogen in response to a nutrient impulse in soil

Fluctuations of bacterial populations can be observed when frequent and sufficiently long series of samples are obtained for direct microscopic or plate counts of bacteria. Fluctuations in bacterial numbers are especially noticeable after some disturbance of soil such as substrate addition. However, very seldom were bacterial fluctuations subjected to proper statistical analysis to detect significant periodical components in the data. In this thesis, bacterial dynamics and distributions were investigated in short-term (1 month) controlled experiments for rhizosphere and bulk soil after substrate input from plant roots and fresh plant debris, respectively. By using harmonics analysis, significant wave-like oscillations in bacterial numbers were detected along wheat roots and over time after clover-grass incorporation into soil. Oscillations were not related to numbers of lateral roots, soil moisture, mineral nitrogen concentrations, pH or redox potential. Soil fauna was not monitored in the rhizosphere experiments. In clover-grass experiments, nematode populations and composition were monitored daily, while protozoa were enumerated occasionally. Bacterial feeding nematode populations did not oscillate, but daily changes in their numbers did oscillate with a frequency similar to that of bacterial oscillations. Short-term oscillating dynamics of bacterial populations were simulated in two mechanistic models: "BACWAVE" to simulate the wave-like distribution of copiotrophic and oligotrophic bacterial populations along roots and "BACWAVE-WEB" to simulate wave-like oscillations of 3 trophic groups of bacteria (copiotrophic, hydrolytic, and oligotrophic) and their predators (protozoa and nematodes) over time after incorporation of plant residues into soil. In both models relative growth and death rates were non-linearly related to readily utilizable substrate concentrations, so that the cross-over point of the growth and death rate curves occurred at realistic substrate concentrations. A good fit was obtained to the observed data. Mineral nitrogen release was also modeled satisfactorily with "BACWAVE-WEB". Virtual experiments suggested that substrate-bacteria interactions are more important for regulation of bacterial oscillations than predator-prey interactions, and that regulation by protozoa was more influential than that by nematodes. Release of mineral nitrogen was primarily determined by bacteria, and to a lesser extent by predators. Oligotrophic bacteria played an important role in stimulating hydrolytic enzyme production and moderating bacterial fluctuations. T<span style='letter-spacing:-.1pt'>he "BACWAVE-WEB" model has good potential to predict responses of microbial communities to a disturbance, which could be used to characterize soil health</span>.</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;mso-ansi-language:EN-GB'><o:p></o:p></span><strong><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPS-BoldMT; mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:TimesNewRomanPS-BoldMT; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'><br clear=all style='page-break-before:always'></span></strong><p class=MsoBodyText align=center style='text-align:center;line-height:130%'><strong><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPS-BoldMT; mso-bidi-font-family:TimesNewRomanPS-BoldMT;mso-ansi-language:EN-GB'>Summary<o:p></o:p></span></strong><p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><strong><span lang=EN-GB style='font-size:11.0pt;font-family:TimesNewRomanPS-BoldMT; mso-bidi-font-family:TimesNewRomanPS-BoldMT;mso-ansi-language:EN-GB'><o:p> </o:p></span></strong><p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><strong><span lang=EN-GB style='font-size:11.0pt;font-family:TimesNewRomanPS-BoldMT; mso-bidi-font-family:TimesNewRomanPS-BoldMT;mso-ansi-language:EN-GB'><o:p> </o:p></span></strong><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>Fluctuations of bacterial populations can be observed when frequent and sufficiently long series of samples are obtained for direct microscopic or plate counts of bacteria. Such fluctuations in time and space have been observed for both bacteria and other soil inhabitants. These fluctuations of bacterial numbers are especially noticeable after some disturbance of soil such as tillage, drying and rewetting, and substrate addition, for example in the form of fresh plant material. However, very seldom were bacterial fluctuations subjected to proper statistical analysis to detect significant periodical components in the analyzed data (Chapter 1). The phenomenon of wave-like bacterial oscillations was investigated in short-term (1 month) controlled experiments for rhizosphere and bulk soil after substrate input from plant roots and fresh plant debris, respectively. Short-term oscillating dynamics of bacterial populations were simulated in a mechanistic model, which may contribute significantly to our understanding of the reasons and consequences of bacterial oscillations after addition of substrate to soil.<o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>To determine the spatial variation in density of different trophic bacterial groups (copiotrophic and oligotrophic) and carbon sources in the rhizosphere, colony-forming units (CFUs) and soluble total organic carbon (TOC) were quantified along the root from rhizosphere and corresponding bulk soil samples at 2 cm intervals along wheat roots two, three, and four weeks after planting (Chapter 2). There was a moderate rhizosphere effect in one experiment with soil rich in fresh plant debris (1% C in soil), and a very pronounced rhizosphere effect in the second experiment with soil low in organic matter (0.7% C). Wave-like patterns of both trophic groups of bacteria as well as TOC could be discerned along the whole root length (60 or 90 cm). Harmonical analysis revealed significant oscillations in bacterial populations and TOC. TOC concentrations were maximal at the root tip and base and minimal in the middle part of the roots. Populations of copiotrophic and oligotrophic bacteria had two maxima close to the root tip and at the root base, or three maxima close to the tip, in the middle section, and at the root base. Phases and periods of the two trophic groups differed slightly. The location and pattern of the waves in bacterial populations changed progressively from week to week, and was not consistently correlated with TOC concentrations or the location of lateral root formation. Thus, the traditional view that patterns in bacterial numbers along the root directly reflect patterns in exudation and rhizodeposition from several fixed sources along the root may not be true. We attributed the observed wave-like patterns in bacterial populations to bacterial growth and death cycles (due to autolysis or grazing by predators). Considering the root tip as a moving nutrient source, temporal oscillations in bacterial populations at any location where the root tip passed would result in moving waves along the root. This change in concept about bacterial populations in the rhizosphere could have significant implications for plant growth promotion and soil health.<o:p></o:p></span><p class=MsoNormal style='text-align:justify;mso-layout-grid-align:none; text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>To check the hypothesis that the principal mechanism underlying the wave-like distribution of bacteria along the root is a cycle of growth, death, autolysis, and regrowth of copiotrophic bacteria in response to a moving substrate source (root tip) a simulation model was created (Chapter 3). After transformation of observed spatial data to presumed temporal data based on root growth rates, a simulation model was constructed with the Runge-Kutta integration method to simulate the dynamics of colony-forming bacterial biomass, with relative growth and death rates depending on substrate content so that the rate curves crossed over at a substrate concentration within the range of substrate availability. The original source of substrate was the root tip, supplemented with a background flux (BGF) of substrate from soil organic matter and dead root cells. Dead necromass from bacteria was partially recycled into substrate. This model was named "BACWAVE", standing for 'bacterial waves'. The model generated cyclic dynamics of bacteria, which were translated into traveling spatial waves along a moving nutrient source. Parameter values were estimated from calculated initial substrate concentrations and observed microbial distributions along wheat roots by an iterative optimization method. The kinetic parameter estimates fell in the range of values reported in the literature. The model was validated with an independent data set of bacteria along wheat roots in relatively C-rich soil. Calculated microbial biomass values produced spatial fluctuations similar to those obtained for experimental biomass data derived from colony forming units. Concentrations of readily utilizable substrate (RUS) calculated from biomass dynamics did not mimic measured concentrations of TOC, which consists not only of substrate but also various polymers and humic acids. Thus, a moving impulse of nutrients into soil resulting in cycles of growth and death of bacteria can explain the observed phenomenon of moving bacterial waves along roots. This was the first report of wave-like dynamics of micro-organisms in soil along a root resulting from the interaction of a single organism group with its substrate.<o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>The model "BACWAVE" for wave-like dynamics of copiotrophic bacteria (CB) was extended to include dynamics of oligotrophic bacteria (</span><st1:place><span lang=EN-GB style='font-size:11.0pt; line-height:130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>OB</span></st1:place><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>) (Chapter 4). CFUs of</span><st1:place><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>OB</span></st1:place><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>and CB along wheat roots (24 samples) in a low C soil were transformed to temporal biomass taking root growth rate and cell sizes into account. Growth rates of both groups of bacteria increased with readily utilizable substrate (RUS) according to Monod equations, but each with their own characteristic parameter values. The death rate of CB decreased monotonically with substrate concentration, while the death rate of</span><st1:place><span lang=EN-GB style='font-size: 11.0pt;line-height:130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family: TimesNewRomanPSMT;mso-ansi-language:EN-GB'>OB</span></st1:place><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>first decreased and then increased with substrate concentration. Model parameters were estimated from literature and with an iterative optimization method. Initial biomass and kinetic parameters were lower for</span><st1:place><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>OB</span></st1:place><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>than for CB, and fell in the range of values in the literature. The model was validated with an independent data set of bacteria along wheat roots in relatively C-rich soil, so that BGF and initial microbial populations were higher, but other model parameters were the same for both data sets. A satisfactory fit was obtained between experimental and modeled data. This is the first rhizosphere model in which oligotrophic bacteria are taken into account.</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;mso-ansi-language:EN-GB'><o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>Several microcosm experiments were carried out to investigate the hypothesis that an impulse of fresh substrate into soil would invoke oscillations in bacterial populations (Chapter 5). Soil bacterial populations, mineral nitrogen content, pH, and redox potential (ROP) were monitored daily for one month after incorporation of clover-grass (CG) plant material in soil. Colony-forming units (CFUs) and direct microscopic counts of FDA-stained and FITC-stained bacteria increased immediately after incorporation of the plant material, dropped within 2 days, and fluctuated thereafter. Harmonics analysis demonstrated that there were significant wave-like fluctuations with three or four significant peaks within one month after incorporation of clover-grass material. Ammonium (N</span><span lang=EN-US style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-US'>H ₄⁺ )</span><span lang=EN-US style='font-size:11.0pt;line-height:130%;mso-ansi-language:EN-US'></span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>concentrations increased from the start of the experiments until nitrification commenced. Nitrate (NO ₃</span>^{<span lang=EN-GB style='font-size:11.0pt; line-height:130%;mso-ansi-language:EN-GB'>−</span>}<span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>) concentrations dropped immediately after plant incorporation, and then rose monotonically until the end of the experiments. There were no wave-like fluctuations in N</span><span lang=EN-US style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-US'>H ₄⁺</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>and NO ₃</span>^{<span lang=EN-GB style='font-size:11.0pt;line-height:130%;mso-ansi-language:EN-GB'>−</span>}<span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>concentrations, so that bacterial fluctuations could not be attributed to alternating mineral N shortage and sufficiency. pH levels rose and declined with N</span><span lang=EN-US style='font-size:11.0pt;line-height:130%; font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-US'>H ₄⁺</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>levels. ROP dropped shortly before N</span><span lang=EN-US style='font-size:11.0pt; line-height:130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-US'>H ₄⁺</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>concentrations rose, and increased before N</span><span lang=EN-US style='font-size:11.0pt; line-height:130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-US'>H ₄⁺</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;font-family:TimesNewRomanPSMT; mso-bidi-font-family:TimesNewRomanPSMT;mso-ansi-language:EN-GB'>concentrations decreased; there were no regular fluctuations in ROP, so that temporary oxygen shortages may not have been responsible for the observed fluctuations in bacterial populations. Thus, for the first time, regular wave-like dynamics were demonstrated for bacterial populations after perturbation by addition of fresh organic matter to soil, and several potential reasons for the death phase of the fluctuations could be excluded from further consideration.<o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>To elucidate possible reasons for the oscillations in bacterial populations, potential interactions with populations of bacterial predators, in particular bacterial-feeding nematodes (BFN), were investigated (Chapter 6). In two microcosm experiments, soil bacteria (CFU's and microscopic counts of stained bacteria) and nematode populations in 22 families were monitored daily for 25 or 30 days after incorporation of clover+grass (CG) plant material into soil. Soil bacterial populations fluctuated significantly after incorporation of the plant material with 2 peaks within the first week and 3 or 4 smaller peaks thereafter. Total nematodes and BFN populations started to increase in the course of the second week after CG incorporation, but the proportion of BFN increased within one week. Inactive juvenile BFN (dauerlarvae) seemed to be activated after two days (as the percentage of Rhabditidae increased and dauerlarvae decreased), followed by step-wise increases in dauerlarvae every four days, indicating that there was a new generation every four days. There were significant wave-like fluctuations in daily population changes of BFN, but not in total nematode communities, over the duration of these experiments. These fluctuations had similar periods (5 days) as those of bacterial populations, but were shifted about 3 days relatively to the bacterial fluctuations.</span><span lang=EN-GB style='font-size:11.0pt;line-height:130%;mso-ansi-language:EN-GB'><o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;mso-ansi-language:EN-GB'><o:p> </o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%;mso-layout-grid-align: none;text-autospace:none'><span lang=EN-GB style='font-size:11.0pt;line-height: 130%;font-family:TimesNewRomanPSMT;mso-bidi-font-family:TimesNewRomanPSMT; mso-ansi-language:EN-GB'>In another microcosm experiment, dynamics of bacterial populations were monitored in response to gamma-irradiated plant material added to gamma-irradiated soil mixed with filtered bacterial suspensions and to non-irradiated soil. Gamma-irradiation of soil significantly increased the periods and amplitudes of bacterial oscillations compared to untreated field soil. Nematode populations were decimated in gamma-irradiated soils, but a small number of protozoa were accidentally introduced in the irradiated soil, and may have been partially responsible for the delayed regulation of bacterial growth. We conclude that fluctuations in bacterial populations were not directly