archives@tulane.edu / Mounting evidence suggests that microbial symbionts can play a crucial role in promoting plant invasion. Still, ecologists lack a complete understanding of how microbially-mediated invasion mechanisms operate across different abiotic and biotic contexts. In this dissertation, I characterize the microbial communities associated with and invasive grass, Phragmites australis, and native marsh species in coastal Louisiana, and examine how saltwater intrusion may differentially alter microbially-mediated interactions among these species. In a field survey, I found that P. australis associates with root and soil fungal communities with higher richness, diversity, and pathogen abundances compared to native species; however, I found no evidence of compositional changes or pathogen spillover in native species in close proximity to the invasion front. Additionally, in a greenhouse mesocosm experiment, I found that salinity-induced changes in microbial communities may enhance P. australis invasion in freshwater wetlands, but the magnitude of these effects depended on plant community context. Finally, using a plant-soil feedback experiment, I found that salinity and microbes adapted to salinity synergistically promote native coexistence in native communities, but may facilitate invasive dominance in invaded communities. This dissertation demonstrates that outcomes of plant-microbe interactions may shift across heterogenous abiotic and biotic landscapes. Furthermore, results presented here may help in predicting how saltwater intrusion will influence microbially-mediated invasion dynamics as global change progresses. / 1 / Carolyn Schroeder

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This study uses scanning electron microscopy (SEM) to examine the occurrence of bacteria and their exopolysaccharide slime layer in microbial enhanced oil recovery experiments. A test of SEM preservation techniques showed that air drying and 10% glutaraldehyde fixation preserved the slime layer but distorted and flattened bacteria. Techniques with ethanol dehydration preserved the bacterial textures but fragmented the slime layer. In sandstones that had been plugged during microbial enhanced oil recovery experiments, bacteria are sparsely distributed. An irregular, confluent slime sheet covers grains and coats pore spaces and is responsible for permeability modification in microbial enhanced oil recovery. The development of the slime layer over time involves several steps: growth of ultramicrobacteria into full-sized bacteria; creation of a slime capsule; growth of globular masses, ropy masses, webs, thin sheets; and growth of a thicker, poreilling mass of slime associated with large balls of slime.

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To understand the soil microbial community structures of different locations and altitudes, as well as the fluctuation of microbial populations foward seasonal change, this study investigated the biodiversity of soil microbiota of eight sampling sites along the Lao-Nong River basin during 4/11/2003~3/21/2004. The results showed that the soil properties were different among these sampling sites. The soil temperatures were ranging in between 24~32¢J, except Tianchr, the highest altitude sampling site (7~19¢J) ; pH values were 4.00 (Tianchr) ~8.20 (Tauyuan) ; moisture contents were 2.31% (Liouguei) ~70.23% (Tianchr) ; total organic carbon contents were 0.38% (Linkou) ~36.21% (Tianchr) ; total organic nitrogen contents were 0.067% (Tauyuan) ~0.672% (Tianchr); the C/N ratio were 7.0~25.0, except Tianchr (42 in average) . In microbial community structures analysis, bacteria were the most abundant organisms among all soil samples (106~108 CFU per gram of soil) . The amount of other microbes were as follow: nitrogen-fixing microbes 105~107 CFU, actinomycetes 104~107 CFU, fungi 104~106 CFU, cellulolytic microbes 104~106 CFU, and the least one was phosphate- solubilizing microbes 103~105 CFU. For molecular biological microbiota analysis, the bead-beating DNA extraction method resulted the richest bacterial 16S rDNA DGGE profile. The16S rDNA DGGE profiles of various samples taken from different seasons showed somewhat seasonal variations that might relate to the specific climates of Taiwan, such as the plum rains and typhoons. Moreover, we also successfully cultured and preserved many microbes with specific biological functions. These microorganisms will provide a useful resource for further research and development in the biotechnology industries.

Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Biochar (charcoal) is lauded by many scientists as an effective way to remove carbon dioxide from the atmosphere and storing it in a very stable form in the soil for hundreds to thousands of years, whilst promoting soil fertility and productivity. Considering that no significant amounts of charcoal are presently accumulating in the environment, despite considerable amounts produced globally in natural and man-made fires, this study focuses on understanding the degradation of biochar and its interactions with plants and soil organisms. The following experiments were conducted to achieve this goal. Controlled chemical oxidation of biochar, using different concentrations of hydrogen peroxide, was conducted in an attempt to mimic the enzymatic degradation of biochar by basidiomycetes. The changes occurring in biochars structure and chemistry were assessed afterwards. Furthermore, aerobic and anaerobic digestion of biochar was conducted in vitro, and in vivo to investigate the changes occurring in biochar‘s elemental composition and chemistry during oxidation and factors that play a determining role in the rate of biochar degradation. The influence of biochar in soil on free-living and symbiotic microbial communities as well as its impact on total plant biomass production and root development was assessed in three greenhouse pot trials using wheat and green beans as test plants It was proven that biochar is almost fully H2O2-degradable, mostly through hydroxylation and carboxylation reactions which led to the formation of various short chained carboxylic acids, surface saturation with acidic functional groups as determined by the surface acidity measurements and proven by the increase in the intensity of FT-IR peaks associated with carboxyl and phenolic C-O groups. Furthermore, hydrogen peroxide treatment resulted in preferential removal of volatile organic carbons and led to the purification of biochar as evident by the new, more intense and sharper peaks in the region of 1600-1000 cm-1. These FT-IR peaks are considered as the more recalcitrant fraction of biochar and were shown to be mostly associated with transformation products of lignin and cellulose formed during pyrolysis. The incubation trial confirmed that biochar cannot be utilized as a sole carbon source without the addition of nutrients or glucose, to activate microbial activity within the columns. Furthermore, abiotic oxidation can be facilitated by oxidative soil minerals such as birnessite, but oxidation with atmospheric oxygen did not result in the evolution of CO2 from biochar. The average CO2 production in pot trials without plants in both the fertilized and unfertilized treatments increased linearly (R2= 0.80; 0.79 respectively) with increasing biochar application rates when biochar was the main carbon sources. Anaerobic degradation of biochar by a methanogenic consortium was much more efficient in utilizing biochar as a carbon source, compared to aerobic digestion. The anaerobic digesters maintained a chemical oxygen demand (COD) removal efficiency of 30% per week with continuous production of CO2, whilst methane production was very erratic. We proposed that better control over pH and alkalinity as well as an increase in hydraulic retention time would improve both the COD removal efficiency and methane production. Field incubations resulted in various degrees of oxidation at different incubation sites. An increase in the oxygen content and a decreased in the carbon content of biochar‘s elemental composition and also an increase in the surface acidity due to a larger amount of carboxyl acid groups on the surface as seen in the increase in the FT-IR peak at 1700 cm-1 confirmed that biochar are susceptible to oxidation under field conditions. We came to the conclusion that oxidation and mineralization of biochar in this trial occurred at a faster rate in soils with a higher microbial activity. The pot trials, confirmed that biochar does not serve as a fertilizer even though it did increase total biomass production between biochar application rates of 0.05-2.5 % (w/w). For agricultural purposes the addition of biochar should always be applied together with NPK fertilizer. In both the wheat and green bean trials it was confirmed that biochar application rates of 0.05-0.5% (w/w) on the sandy, slightly acidic soil used in this trial resulted in the greatest biomass production and fertilizer use efficiency. Biochar additions resulted in considerable increases in soil pH and C/N ratios which were considered as the main reasons for the decrease in microbial biomass in the unfertilized green bean treatments as it made the uptake of N more limited. The addition of fertilizer however, alleviated N-supply constraints and as a result promoted microbial growth at all biochar application rates of pot trial 1. However, biochar did not promote mycorrhyzal colonization and caused a decrease in the mycorrhizal colonization of roots with increasing biochar application rates and within biochar layers. Biological nitrogen fixation, however, reacted positively to the addition of biochar. High biochar application rates significantly enhanced the plants reliance on these symbiotic relationships. We hypothesized that biochar physically immobilized N into its microvoids through capillary suction and then served as a physical barrier between plant roots and absorbed N. However, immobilzation of N by microbes could also have contributed to the decrease in N uptake if one takes into account that microbial activity was higher (respiration data) at the higher biochar application rates. Further investigations are needed to warrant this hypothesizes. / AFRIKAANSE OPSOMMING: Biochar (houtskool) is deur talle wetenskaplikes die lof toegeswaai as ‘n doeltreffende manier om koolstofdioksied uit die atmosfeer te verwyder en in ‘n baie stabiele vorm in die grond vir honderde tot duisende jare te stoor, terwyl dit die grondvrugbaarheid en produktiwiteit bevorder. As daar in ag geneem word dat geen beduidende hoeveelheid houtskool in die omgewing opgaar nie ondanks groot hoeveelhede wat wêreldwyd deur natuurlike en mensgemaakte brande gevorm word, is die doel van hierdie studie om die afbraak en die interaksie van biochar met plante en grondmikrobes beter te verstaan. Om hierdie doel te bereik is die volgende eksperimente uitgevoer: Beheerde chemiese oksidasie is op die biochar toegepas deur gebruik te maak van verskillende konsentrasies waterstofperoksied in 'n poging om die ensiematiese afbraak van biochar deur basidiomysete na te maak. Die veranderinge wat plaasvind in die struktuur en chemie van biochar is daarna bestudeer. Daarbenewens is die aerobiese and anearobiese afbraak van biochar toegepas beide in vitro- en in vivo-, om die veranderinge wat in biochar se elementele samestelling en chemie plaasvind gedurende oksidasie en ook die faktore wat 'n bepalende rol in die tempo waarteen biochar afbreek, te ondersoek. Die invloed van biochar in die grond op vrylewende en simbiotiese mikrobiese populasies, sowel as die impak daarvan op die totale plant biomassa produksie en ontwikkeling van plantwortels, is vasgestel tydens drie groeitonnel potproewe waarby koring en boontjies as planttoetsspesies gebruik is Dit is bewys dat biochar byna volledig deur H2O2 afgebreek kan word, meestal deur hidroksilasie en karboksilasie reaksies wat gelei het tot die vorming van 'n verskeidenheid kort ketting karboksielsure, 'n biochar oppervlak versadig met suurvormende funksionele groepe soos bepaal en bewys deur die toename in intensiteit van die FT-IR (Fourier Transvorm Infrarooi Spektroskopie) pieke geassosieer met karboksiel en fenoliese C-O groepe. Die behandeling van biochar met H2O2 het by voorkeur die vlugtige organise koolstof verwyder wat gelei het tot suiwering van die biochar, wat bevestig is deur die nuwe, meer intens en skerper FT-IR pieke in die area tussen 1600-1000 cm-1. Die FT-IR pieke word beskou as die meer weerstandbiedende fraksie van biochar en daar is bewys dat die pieke meestal met getransformeerde produkte van lignien en sellulose wat tydens pirolise gevorm is, geassosieer word. Die inkubasie proef het bevestig dat biochar nie deur mikrobes benut kan word as enigste bron van koolstof sonder die byvoeging van nutriente of glukose nie, om die mikrobes binne die inkubasie kolom te aktiveer. Daarbenewens kan abiotiese oksidasie van biochar deur oksidatiewe grondminerale soos birnessite (δ-MnO2) gefasiliteer word, terwyl oksidasie van biochar deur atmosferiese suurstof nie tot enige CO2 produksie gelei het nie. Nogtans het die gemiddelde CO2 produksie in die boontjie potproef, sonder die plante, in beide die onbemeste en bemeste behandelings linieer toegeneem (R2= 0.80; 0.79 onderskeidelik) met toenemende aanwendingskoers van biochar, toe biochar die dominante bron van koolstof was. Anaerobiese afbraak van biochar deur 'n metanogeniese konsortium was heelwat meer effektief in die benutting van biochar as enigste koolstofbron in vergelyking met aerobiese afbraak. Die anaerobiese verteertoestel het konstant 30% van die chemiese suurstof behoefte (CSB) weekliks verwyder, gepaardegaande met die voortdurende produksie van CO2, terwyl metaangasproduksie baie onegalig was. Dit word voorgestel dat met beter beheer oor pH en alkaliniteit en ook 'n langer hidrouliese retensie tyd, kan beide die CSB verwyderingseffektiwiteit en metaangasproduksie verbeter kan word. Veld inkubasies het verskeie mates van oksidasie meegebring tussen die verskillende inkubasie liggings. 'n Toename in die suurstofinhoud en 'n afname in die koolstof inhoud van biochar se elementele samestelling sowel as 'n toename in die oppervlak suurheid weens die groter hoeveelheid karboksielsure aan die oppervlak soos blyk uit die FT-IR piek by 1700 cm-1, het bevestig dat biochar wel vatbaar is vir oksidasie onder veld kondisies. Die gevolgtrekking was dat biochar oksidasie en mineralisasie in hierdie proef teen 'n vinniger tempo plaasgevind het in die gronde met hoer mikrobiese aktiwiteit. Die potproewe het bevestig dat biochar nie as bemestingsstof sal dien nie, alhoewel dit tot 'n toename in die biomassa produksie gelei het tussen die biochar aanwendingskoerse van 0.05-2.5% (w/w). Vir landbou doeleindes moet die aanwending van biochar altyd gepaardgaan met NPK bemesting. Beide die koring- en boontjie proewe het bevestig dat die biochar aanwendingskoerse tussen 0.05-0.5% (w/w) op die sanderig, effens suur grond wat gebruik is in die proef, gelei het tot die hoogste biomassa produksie en bemestingseffektiwiteit. Die toediening van biochar het gelei tot merkbare toenames in grond pH en C/N verhoudings en hierdie toestande was beskou as die hoof redes vir die afname in mikrobiese biomassa in die onbemeste boontjie behandelings omdat dit die opname van N meer beperk. Die toediening van bemesting het egter die beperkings op N voorsiening opgehef en as gevolg hiervan die mikrobiese biomassa bevorder by alle biochar aanwendingskoerse. Biochar het egter nie mikorrisa kolonisasie bevorder nie en het gelei tot =n afname in die mikorrisa kolonisasie van die wortels met toenemende biochar aanwendingskoerse en binne in die biochar lae van potproef 1. Biologiese stikstof vaslegging het egter positief reageer op die toediening van biochar. Hoë biochar aanwendingskoerse het beduidend die plant se afhanklikheid op hierdie simbiotiese verhouding verhoog. Ons hipotese is dat die biochar fisies N immobiliseer binne in die mikro-ruimtes deur kapillêre suigaksie en dan as 'n fisiese versperring dien tussen die plantwortels en die geabsorbeerde N. Die immobilisasie van minerale N deur mikrobes kon egter ook grootliks bygedra het tot die afname in N opname as daar in ag geneem word dat mikrobiese aktiwiteit (respirasie data) hoër was by die hoër biochar aanwendingskoerse. Verdere ondersoeke moet daarom uitgevoer word om hierdie hipotese te bevestig.

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Microbes rely on collective behaviours, such as communication and cooperation to survive and form communities. The majority of these social behaviours are mediated by the secretion of public good molecules into a shared environment such that they can be utilized by neighbouring cells. Therefore, individuals that engage in costly cooperative behaviour are susceptible to exploitation by selfish cheats that gain the benefit of cooperation without investing their share of the public good cost. Understanding such bacterial social interactions and the underlying molecular mechanisms gives insight into their complex social life in natural environments and can be used to develop alternative treatments for pathogenic bacteria that rely on such social interactions for virulence and to infect hosts. In this thesis I examine social behaviours expressed by the opportunistic pathogen, Pseudomonas aeruginosa. I develop an understanding of bacterial social dynamics, particularly competitive dynamics between cooperator and cheat strains and strains that engage in bacteriocin-mediated chemical warfare. I investigate bacterial cheat-cooperator systems in several ways: 1) I begin with a review describing the evolution of and response to cheating across a range of organisms and discuss the confusion that arises in identifying cheats particularly in microbial studies and therefore propose a key to identify cheating behaviour. 2) I empirically test whether cheating behaviour is context dependent in bacterial populations and reveal that the ability to cheat varies with the abiotic and social environment, which are two fluctuating conditions in natural environments. 3) I take an experimental approach to investigate why cheat invasion is not commonly observed in natural bacterial populations by testing the effect of cooperative bacterial growth dynamics on cheating ability. I find that secretion of public goods varies with bacterial growth dynamics and physiological growth stages which may explain why cheat invasion is more commonly observed in lab cultures and not in established natural populations. 4) In the final chapter I experimentally use natural isolates to examine the role of bacteriocins in mediating competition in pathogenic populations and find that contrary to empirical and theoretical work, bacteriocins do not play a significant role in strain competitive success and dominance. The thesis has laid groundwork for studying and understanding the role of social behaviours in bacterial systems and for further exploring social dynamics in natural bacterial populations.