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C H A P T E R 7

C H A P T E R 7. Microbial Transport. Outline. 1.INTRODUCTION 2.FACTORS AFFECTING MICROBIALTRANSPORT 3.Microbial Filtration 4.Physiological State. Transport of microbes or of their genetic information is a complex issue of growing concern.

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C H A P T E R 7

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  1. C H A P T E R 7 Microbial Transport

  2. Outline 1.INTRODUCTION 2.FACTORS AFFECTING MICROBIALTRANSPORT 3.Microbial Filtration 4.Physiological State

  3. Transport of microbes or of their genetic information is a complex issue of growing concern. The introduction of microorganisms into an environmental system including enhanced biodegradation of organic contaminants, remediation of metal contaminated sites, improvement of soil structure. Microbial inoculation with the intent of deriving such benefits is referred to as bioaugmentation. Transport: to carry or cause to go from one place to another, esp over some distance. Introduction :addition Remediation: the action of remedying something, esp the reversal or stopping of damage to the environment Augmentation: the act of augmenting or the state of being augmented Augment: to make or become greater in number, amount, strength, etc; increase 7.1 INTRODUCTION

  4. 7.2 FACTORS AFFECTING MICROBIALTRANSPORT • Transport of microorganisms is governed by a variety of abiotic and biotic factors. These factors include the following: • filtration effects, • physiological state of the cells, • Influence of Cell Surface Properties

  5. 7.2.1 Microbial Filtration Most bacteria range from 0.3 to 2μ m in diameter, and micropores can be much smaller in size. As a result, bacterial cells are excluded from the micropores (Fig. 7.1) diameter :the length of a straight line passing through the center of a circle and connecting two points on the circumference

  6. - Bacterium has access (throat • 通道)to micropore • B. - Micropore exclusion: pore is too small for the bacterial cell • C. - Micropore exclusion:pore throat(passage) • is too small to allow cell into pore FIGURE 7.1 Exclusion of a bacterial cell from microporous domains in structured porous media.

  7. There can be detrimental consequences of micropore exclusion in a soil.

  8. when contaminants diffuse into micropores that exclude bacteria, they become unavailable forbiodegradation. Because diffusion occurs slowly, this is a problem that normally worsens(问题加剧) as the contact time between the contaminant and the porous medium increases. This process, known as contaminant aging, results in slower rates of contaminant degradation. 1.unavailable for: not available or accessible 2. diffusion : (physics) the process in which there is movement of a substance from an area of high concentration of that substance to an area of lower ... 3.aging: ripening

  9. (1) residuematter that remains after something has been removed For example, residues of EDB (1,2-dibromoethane) were found to persist in（remain）fields for as long as 19 years after application. (2) 1,2-dibromoethane: 1,2-二溴乙烷； (3) persist in： remain

  10. 7.2.2 Physiological State 1.Exopolymers Exopolymersincrease the effectivediameter of a cell; may adhere to pore surfaces, inessence decreasing pore size; and may modify solidsurfaces to promote attachment. All of thesemay lead to pore clogging.Pore clogging can severely limit bioaugmentation Physiological:relating to the biological study of physiology Clogging：preventing movement Modify:make less severe or harsh or extreme

  11. Starvation is an important in movement of soil organisms since nutrients are often limiting in porous media, and thus a large proportion of the bacteria are in starved or semistarved states. In 1981, Torrella and Morita coined the term(define) ultramicrobacteria to describe bacteria that were less than 0.3μm in diameter.These small bacteria demonstrated slow growth and did not significantly increase in size when inoculated onto a nutrient-rich agar（any culture medium that uses agar as the gelling agent）medium. 2.starvation Under starvation conditions, bacteria decrease in size to 0.3 μm or even smaller and shed(get rid of) their glycocalyx or capsule layer. This may increase their transport potential, because both cell size and surface properties are changed.

  12. Ultramicrobacteria not only occur in natural environments but also can be created in the laboratory by placing normal cells under starvation conditions. It is thought that a large proportion of environmental isolates（bacteria）can be starved into ultramicrobacteria and then resuscitated successfully.  to restore to consciousness

  13. Herman and Costerton subjected（做实验）a p-nitrophenol(对-硝基苯酚) degrader（bacteria）isolated from a waste lagoon to starvation by placing it in phosphate-buffered saline for 10 weeks.The difference in cell size and morphology before and after starvation is clearly shown in Fig. 7.2A and B.The starved cells were then resuscitated by adding p-nitrophenol to the medium (Fig. 7.2C and D). Lagoon： a body of water cut off from a larger body by a reef of sand or coral Saline： an isotonic solution of sodium chloride and distilled water morphology:dealswith the structure of living things

  14. FIGURE 7.2 (A) Phase-contrast micrograph（相差显微照片） of an isolate grown on p-nitrophenol as the sole carbon source. (B) Phase-contrast micrograph of the p-nitrophenol degrader after 10 weeks of starvation in phosphate-buffered saline. (C) Electron micrograph of the starved p-nitrophenol degrading isolate. (D) Electron micrograph of the starved p-nitrophenol-degrading isolate after resuscitation on p-nitrophenol.

  15. To demonstrate the difference in transport of ultramicrobacteria and normal cells, MacLeod et al. examined the movement of starved and normal Klebsiella pneumoniae （肺炎克雷伯氏菌）cells through glass bead（空心玻璃珠）columns. The starved cells penetrated further into the column（Figure 7.3）.

  16. FIGURE 7.3 The different permeability reduction profiles of fused glass bead cores injected with K. pneumoniae cells either in a vegetative state or starved for a period of time in phosphate buffered saline. The starved cells penetrated further into the column than did the vegetative cells. Permeability：the property of something that can be pervaded by a liquid (as by osmosis or diffusion)

  17. Starved cells were evenly distributed throughout the column, whereas the vegetative cells were found in much higher numbers near the inlet end of the column (Fig. 7.4).

  18. FIGURE 7.4 Differences in the DNA-derived cell distribution in cores injected with either a vegetative cell culture of K. pneumoniae or a cell suspension that was starved in phosphate buffered saline for 2 weeks.

  19. Uponnutrient stimulation, the starved cells were found toenter a state of growth accompanied by increasedpolymer production. This work demonstrates（shows） that inoculationwith starved cells followed by nutrientstimulation has potential for increasing bioaugmentationefforts(effects). For example, a starved cell that migratesfarther throughthe terrestrial profile(剖面) has an increasedlikelihood of reaching a targeted(objective,aim) contaminated site.Once at the site, the contaminant can serve as（use as） a nutrientsource, inducing(cause to arise) the microbe to enter a metabolicallyactive state.

  20. 7.2.3 Microbial—The Influence of Cell Surface Properties The key cell surface factors influencing adhesion： 1. Charge； 2.Hydrophobicity(the property of being water-repellent; tending to repel and not absorb water)； 3. The composition of the lipopolysaccharide layer ； 4. The presence of specific proteins in cell surfaces； 5. Appendages,or extracellular polymers .

  21. Once a cell is in the vicinity of a surface, this initial interaction can occur in one of three ways: 1.Diffusionis a result of Brownian motion and allows random interactions of cells with surfaces； 2. Convective transport（对流迁移） is due primarily to water movement and can be several orders of magnitude faster than diffusive transport； 3.Active movement(Fig. 7.5).

  22. FIGURE 7.5 Different ways in which a cell can approach a solid surface.

  23. After initial adhesion, cells can become irreversiblyattached to a particle surface. Irreversible attachmentis a time-dependent process that occurs as a result ofthe interaction of cell surface structures such as fimbriae（菌毛）with the solid surface or as a result of the productionof exopolymers that cement（fasten） the microbial cellto the surface (Fig. 7.7).

  24. FIGURE 7.7 Irreversible attachment is mediated by physical attachment of cells to a surface, which can occur via production of exopolymers or special cell surface structures such as fibrils.

  25. 7.2.9 Persistence and Activity of Introduced Microbes 接入微生物的存活和活性 Typically,there is a rapid decline in numbers of inoculant cells,and there is often a decrease in the average activity per cell of the surviving introduced microbes： 1.Temperature, 2.pH, 3.soil texture, 4.moisture content, 5.the presence of indigenous organisms

  26. 4.moisture content Increases in moisture content above a certain optimal(most desirable) level have actually been shown to lead to a decrease in microbial numbers in a natural soil. This may be due to oxygen depletion(the act of decreasing something markedly) as more pores become water filled.

  27. 5.the presence of indigenous organisms Microbial predators such as protozoa tend to be more active at higher soil moisture contents.The population dynamics between soil bacteria and protozoa resulting from an “ideal” predator–prey relationship is depicted in Fig. 7.17. dynamics :the branch of mechanics concerned with the forces that cause motions of bodies

  28. FIGURE 7.17 An “ideal” prey–predator(被捕食者-捕食者) relationship depicting soil bacterial population density as a function of soil protozoan density.

  29. 7.3 TRANSPORT OF DNA DNA的迁移 A dead or inactivated microbe usually breaks open, releasing its genetic material to the environment. Upon lysis, there is potential for the genetic material to be transported or sorbed to colloids where it can remain protected from degradation. Free or desorbed nucleic acids may be reincorporated into other microbes via transformation.This can result in the expression of genes encoded by these nucleic acids or in the potential transport within the intact recipient cell.

  30. 7.4 NOVEL APPROACHES TO FACILITATE MICROBIAL TRANSPORT 促进微生物迁移的新方法 Novel approaches designed to facilitate microbial transport：. 1.Formation of ultramicrobacteria； 2.Biosurfactants, 3.Gene transfer

  31. 7.4.1 Ultramicrobacteria (超微细菌) It has been known for the past 20 years or so that marine bacteria react to starvation by dividing and shrinking to one third their normal size. Such bacteria are referred to as ultramicrobacteria (UMBs). It has been found that 65% of all isolates obtained from soil can be placed in a nutrientdeprived(lacking adequate food) medium such as phosphate-buffered saline and form UMBs. After several weeks of starvation, a distinct morphological change takes place in these cells. As shown in Fig. 7.2, the cells shrink to approximately 0.3μ m in size and become rounder. They also lose their glycocalyx layer, thereby becoming less sticky. These bacteria can then be resuscitated by providing a carbon source

  32. FIGURE 7.2 (A) Phase-contrast micrograph of an isolate grown on p-nitrophenol as the sole carbon source. (B) Phase-contrast micrograph of the p-nitrophenol degrader after 10 weeks of starvation in phosphate-buffered saline. (C) Electron micrograph of the starved p-nitrophenoldegrading isolate. (D) Electron micrograph of the starved p-nitrophenol-degrading isolate after resuscitation on p-nitrophenol.

  33. The interesting feature of UMBs is that they exhibit several characteristics that enhance their transport: First, they are smaller than normal metabolically active cells and hence are less subject to removal by filtration; Second, the UMB cell surface is less sticky because it has no glycocalyx layer. Such UMBs have been shown to penetrate farther into sandstone cores than their vegetative counterparts.

  34. Interest in UMBs first centered on their potential for use in oil recovery. It has been proposed that local(地下) bacteria could be used to form UMBs and be injected into porous oil-bearing strata（层）, followed by nutrient injection. After nutrient addition,the cells should grow and recover in size. Such growth can create plugging within a geological formation,which would allow subsurface（地下） oil flow to be rechanneled,improving oil recovery（回收）.

  35. 人们对UMB最先的兴趣集中在它回收石油的能力方面。现在，人们已经提出这样的设想：从地下分离出来的细菌能被用于形成UMB ，而后被注射到多孔含油层，然后注入营养物。在加入营养物后，细菌应该生长并恢复其大小。这种生长能在地质构造中造成堵塞，这就使地下石油流被改道，从而促进地下石油的回收。

  36. Another potential use proposed for UMBs is in closure of mine tailing heaps.One of the problems in capping （覆盖）a mine tailing heap is that initial revegetation efforts(植被重建) are often difficult under the low-pH conditions of the mine tailings. The low pH may be intrinsic（of or relating to the essential nature of a thing; inherent）to the mining（选矿） process or driven（caused）by the activity of the sulfur oxidizers, obligate aerobes that create acid mine drainage via the oxidation of sulfur compounds within the tailings （尾矿）. UMB另一个潜在用途是封闭尾矿堆，覆盖尾矿堆的问题之—是在尾矿的低pH条件下植被重建存在着困难。低pH是选矿过程所固有的或被硫氧化者的活动所造成。硫氧化茵是专性好氧菌，能通过氧化尾矿中的硫化合物产生酸矿水。

  37. UMBs may have the potential to form a living biobarrier to prevent oxygen diffusion into the mine tailing heap. A preliminary experiment to evaluate whether this approach is feasible showed that UMBs were able to penetrate farther into the mine tailing material than normal bacteria. In addition, mine tailing effluent characteristics before UMB treatment were pH 3.2 and Eh (redox potential) 150 mV. After UMB treatment the pH was increased to 6.4 and the Eh was decreased to 15 mV. These results suggest that UMBs were able to colonize and form a biobarrier to prevent further acid mine drainage formation (Fig.7.18). This is a critical step in the restoration of mine tailing disposal sites.

  38. 超微细胞有潜力生长形成活性的生物屏障，以防止氧扩散进入尾矿堆。评价这种方法是否可行的初步实验证明，UMB比—般正常细菌更能穿透到更深的矿尾物质。此外，在UMB处理前，尾矿流出液的特征是：PH；3.2和Eh＝150mv。处理后pH增加到6.4，Eh降低到15mv。这些结果说明UMB能繁殖和形成生物屏障以防止酸矿水的进一步形成(图7.18)。这是恢复尾矿处理现场的关键一步。超微细胞有潜力生长形成活性的生物屏障，以防止氧扩散进入尾矿堆。评价这种方法是否可行的初步实验证明，UMB比—般正常细菌更能穿透到更深的矿尾物质。此外，在UMB处理前，尾矿流出液的特征是：PH；3.2和Eh＝150mv。处理后pH增加到6.4，Eh降低到15mv。这些结果说明UMB能繁殖和形成生物屏障以防止酸矿水的进一步形成(图7.18)。这是恢复尾矿处理现场的关键一步。

  39. FIGURE 7.18 The use of ultramicrobacteria in capping a mine tailing disposal site. When oxygen and rainwater can freely penetrate the mine tailing surface, acid mine drainage forms, causing effluents from the waste heap to be characterized by low pH and leached metal (A). After injection and resuscitation of ultramicrobacteria,oxygen is quickly utilized, maintaining anaerobic conditions that suppress the formation of acid mine drainage (B).

  40. 7.4.2 Surfactants Bai et al. (1997) investigated the influence of an anionic monorhamnolipid（单鼠李糖脂）biosurfactant on the transport of three Pseudomonas strains with various hydrophobicities through soil under saturated conditions. Figure7.19 shows the breakthrough（穿透） curves for the most hydrophilic strain at different rhamnolipid concentrations.

  41. FIGURE 7.19 The effect of a rhamnolipid (RL) biosurfactant on transport of a Pseudomonas sp. where Co is the CFU/ml in the influent solution, and C is the CFU/ml in the effluent solution. A pore volume is the amount of liquid it takes to fill all of the soil pores in the column.

  42. In this experiment it was found that the surface charge density of the bacteria did not change in the presence of the rhamnolipid, but the negative surface charge density of the porous medium increased. Thus,reduced bacterial sorption may be due to one of several factors including： 1.An increase in surface charge density caused by rhamnolipid（鼠李糖脂） adsorption; 2.Solubilization of extracellular polymeric glue; 3.Reduced availability of sorption sites on porous surfaces. The advection–dispersion transport model used to interpret these results suggests that the predominant effect of rhamnolipid was to prevent irreversible adsorption of cells.

  43. 7.4.3 Gene Transfer Gene transfer between organisms may occur through conjugation（junction: the state of being joined together）, transduction（(genetics) the process of transfering genetic material from one cell to another by a plasmid or bacteriophage）, or transformation（ Gene donor）. Transfer events such as these may make it possible to distribute genetic information more readily through the soil. Gene transfer events between an introduced organism and indigenous soil recipients has been shown to occur and in some cases this results in increased degradation of a contaminant.

  44. Summary • INTRODUCTION • FACTORS AFFECTING MICROBIALTRANSPORT • Microbial Filtration • Physiological State

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