Metabolic uncoupling ofShewanella oneidensis MR1, under the influence of excess substrate and 3, 3′, 4′, 5-tetrachlorosalicylanilide (TCS

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The dissociation between catabolism and anabolism is generally termed as metabolic uncoupling. Experimentally, metabolic uncoupling is characterized by a reduction in the observed biomass yield. This condition can be brought about by: (a)

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   A  RTICLE Metabolic Uncoupling of  Shewanella oneidensis  MR-1, Under the Influence of Excess Substrate and3, 3 ( , 4 ( , 5-Tetrachlorosalicylanilide (TCS) Gaurav Saini, Brian D. Wood School of Chemical, Biological and Environmental Engineering, Oregon State University,Corvallis, Oregon 97331-2702; telephone: 541-223-2550; fax: 541-737-3099;e-mail: sainig@onid.orst.edu Received 16 August 2007; revision received 16 October 2007; accepted 17 October 2007Published online 30 October 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21702 ABSTRACT:  The dissociation between catabolism and ana-bolism is generally termed as metabolic uncoupling. Experi-mentally, metabolic uncoupling is characterized by areduction in the observed biomass yield. This conditioncan be brought about by: (a) excess substrate (as measuredby   S 0 /  X  0 ), and (b) addition of chemical uncouplers suchas 3, 3 0 , 4 0 , 5-Tetrachlorosalicylanilide (TCS). An empiricalmodel is proposed to quantify the uncoupling effects of bothexcess substrate and uncoupler addition on the microbialcultures. Metabolic uncoupling of   Shewanella oneidensis MR-1, under the influence of excess pyruvate and TCS,hasbeenmodeledusingtheproposedexpression.Thedegreeof uncoupling was measured as a fractional reduction intheoretical maximum observed yield. Excess substrate wasobserved to successively reduce biomass yield as substrateconcentration was increased. In the presence of TCS, con-flicting trends were obtained for number yield and protein yield. This could, in part, be attributed to the observedincrease in cellular protein content upon addition of TCS. Excess substrate conditions dominated uncoupling,as compared to uncoupler addition. However, these twoapproaches were found to have additive effects and could, inconjunction, be employed to control biomass growth duringmicrobial processes such as subsurface bioremediation andactivated sludge treatment.Biotechnol. Bioeng. 2008;99: 1352–1360.  2007 Wiley Periodicals, Inc. KEYWORDS:  uncoupling; excess substrate; TCS; yield;protein;  Shewanella Introduction Microbial processes form the crux of some of the mostcommon environmental engineering applications; examplesinclude water treatment and subsurface remediation. Alarge number of Department of Energy (DOE) sites arecontaminated with heavy metals and radionuclides, some of which might be bioremediated in situ by promoting thegrowth of microorganisms that can use these contaminantsas terminal electron acceptors, which leads to a reduction intheir oxidation state (Lovely et al., 1991; Myers and Nealson,1988; Tiedje, 2002). This is usually accompanied by adecrease in contaminant solubility, which renders themimmobile, a process that is generically termed as  bio-immobilization .In general, microbial applications are based on thebacterial ability to consume organics, metals, and otherwaste products as electron donors or acceptors. This isaccompanied by generation of biomass, an excess of whichcould increase the operational cost and reduce the efficiency of engineered systems, such as municipal wastewatertreatment (Liu and Tay, 2001; Liu, 2003; Mayhew and Stephenson, 1997; Wei et al., 2003) and could lead to  bio-clogging   in in situ bioremediation systems. In such subsur-face systems, the biomass can plug the soil pores, causing areduction in the hydraulic conductivity of the system and aninability for the substrate to be transported effectively frominjection wells (McCarty et al., 1998; Seki et al., 2006; Taylor and Jaffe, 1990). In extreme cases, this lack of control overmicrobial growth leads to a shift in the dominant microbialpopulation (due to a higher consumption rate of thepreferred electron acceptor) and possible failure of thebioremediation scheme (Anderson et al., 2003).It has been observed by a number of researchers thatcertain growth conditions can promote a significant drop inbiomass yield. Senez (1962) observed a roughly 50%reduction in the yield of   Desulfovibrio desulfuricans  (strainBerre S) under some growth conditions. It is now knownthat the presence of excess substrate, addition of organicprotonophores, toxins such as heavy metals and antibiotics,and other thermal or physico-chemical processes can all leadto a partial dissociation between catabolism and anabolism Correspondence to: G. SainiContract grant sponsor: DOEContract grant number: DE-FG03-02ER63353Contract grant sponsor: NSFContract grant number: 0449452-BES 1352  Biotechnology and Bioengineering, Vol. 99, No. 6, April 15, 2008    2007 Wiley Periodicals, Inc.  (Chen et al., 2004; Cook and Russell, 1994; Liu, 1998; Mayhew and Stephenson, 1997; Wei et al., 2003). Thisprocess is generically termed  metabolic uncoupling  , and it ischaracterized by a decrease in the biomass yield.The purposeful promotion of metabolic uncoupling hasbeen extensively studied in the context of activated sludgetreatment schemes, with the goal of controlling andreducing the amount of generated biomass (Liu, 2000;Liu and Tay, 2001; Mayhew and Stephenson, 1997; Strandet al.,1999;Wei et al., 2003;Ye etal., 2003). Although to date metabolic uncoupling has not been explored for use insubsurface bioremediation, the judicious use of proto-nophores or other uncoupling compounds might allow formore complete control of subsurface biomass generationduring bioremediation.Most metabolic uncoupling studies have been restrictedto mixed cultures. These include the development of mathematical models for quantification of biomass yieldand degree of uncoupling (Liu, 1998; Liu and Chen, 1997;Liu et al., 1998; Pirt, 1982; Tsai and Lee, 1990; Zeng and Decker, 1995). There is a lack of literature on metabolicuncoupling of pure cultures, although many of them havebeen shown to be very useful for in situ remediationactivities.  Shewanella oneidensis  MR-1 is one such bacteriathat has been widely used as a model organism for inducingmetal reduction (Kostka et al., 1996; Middleton et al., 2003;Myers and Nealson, 1988; Tiedje, 2002).Any microbial process is difficult to employ in the realworld unless it is accompanied by a quantitative processmodel. Existing models for metabolic uncoupling describeeither (1) the effect of excess substrate (Liu, 1996), or (2) theeffect of a chemical uncoupler (Liu, 2000). However, nomodel currently explains the behavior of microbes underthe influence of both excess substrate conditions and thepresence of a chemical uncoupler.This study aims to demonstrate that excess substrate andchemical uncoupler addition can be used judiciously tocontrol the biomass growth of a pure culture, and that thiseffect can be simulated by an empirical model. We conductedbatch experiments using wild-type  S. oneidensis  MR-1, grownwith pyruvate as the sole energy/carbon substrate, underaerobic conditions. Excess substrate conditions, as definedin this study, refer to growth conditions (measured in termsof   S 0 /  X  0 ) that lead to an observed biomass yield which isless than the theoretical maximum value. The biomass yield(ratio of biomass growth to substrate utilized) was modeledas a function of relative initial substrate concentration( S 0 /  X  0 ) and/or relative uncoupler concentration ( C  u /  X  ).Biomass growth was quantified in terms of either cellnumbers (cells/mL) or cell protein content (ppm protein).Correspondingly, the biomass yield was termed as ‘‘number yield’’ and ‘‘protein yield.’’ The use of two different biomassmeasures allows the simultaneous measurement of quanti-tative (biomass yield) as well as qualitative (changes in cellphysiology) effects of metabolic uncoupling. The degree of uncoupling was quantified as the ratio of fractionalreduction in the biomass yield to the theoretical maximumobserved yield (( Y  obs ) max  ). 3, 3 0 , 4 0 , 5-Tetrachlorosalicyla-nilide (TCS), a component in the formulation of soaps,rinses, and shampoos, was used as the model uncoupler inthe current work. It belongs to a class of chemicals termedas protonophores because of their ability to shuttle electronsacross the cell membrane. A number of previous studieshave shown TCS to be capable of reducing the biomass yieldof activated sludge cultures, effectively at lower concentra-tions compared to other uncouplers (Chen et al., 2000, 2002, 2004; Ye and Li, 2005; Ye et al., 2003). Initial experimentation was focused on quantifying andmodeling the effect of excess substrate conditions onbiomass yield. Later, experiments were conducted to assessthe combined uncoupling effect of excess substrate andchemical uncoupler on the biomass yield of   S. oneidensis MR-1 cells. The results of this research are expected tocontribute to a better understanding of cellular metabolismcontrol, by promotion of metabolic uncoupling, whichmight help in successful application of microbial processesin engineered systems. Materials and Methods Bacterial Cultivation and Sampling Procedures A pure culture of facultative anaerobe  S. oneidensis  MR-1,wild-type was used for this study. This proteobacterium wasfirst isolated from the anaerobic sediments obtained fromLake Oneida, NY in 1988 (Myers and Nealson, 1988). Thegenome of this bacterium has been fully sequenced andannotated (Heidelberg et al., 2002), making it an attractiveorganism for microbiological studies. The frozen stock forthe current study was provided by Pacific NorthwestNational Laboratories (PNNL), Richland, Washington. Forexperimentation, the stock culture was prepared by growingcells in 10% Tryptic soy broth (TSB) solution (3 gm/L) indistilled water, for 24 h. The cells were harvested and mixedwith glycerin in 2:1 ratio and stored at   75 8 C. All theuncoupling experiments were conducted using a modifiedversion of minimal growth media suggested by Kostka andNealson (1998), with pyruvate as the sole carbon/energy source. The medium was adjusted to a pH of about 7 using1 molar sodium hydroxide solution.After growth from the stock, the inoculum was diluted in1:100 ratio in 250 mL growth media. All the cultures wereconstantly agitated in a Barnstead Max  Q 4000 incubator,maintained at 30 8 C. Aerobic conditions were maintainedby continuous aeration and Resazurin (1 ppm) acted asthe indicator of dissolved oxygen levels. Samples fromuncoupling experiments were taken at pre-selected intervalsand were divided into three parts. One part of thesamples was filter sterilized using 0.1 m m Acrodisc 1 syringefilters (Pall Life Sciences, Ann Arbor, MI) for determiningthe substrate consumption by High Performance LiquidChromatography (HPLC). The second portion was stainedby 4 0 , 6-diamidino-2-phenylindole (DAPI) and used for Saini and Wood: Metabolic Uncoupling of   Shewanella  1353 Biotechnology and Bioengineering  direct cell enumeration by Epi-fluorescence microscopy (Olympus, Center Valley, PA) at a total resolution of 1,000  using an Olympus 1 UPLFL 100   objective. Black poly-carbonate filters 25mm indiameter witha 0.22 m mpore size(Osmonics, Inc. Minnetonka, MN) were used for micro-scope-slide preparation. The third part of the samples wasused to determine the total protein content of cultures usingMicro BCA TM Protein Assay Kits (Pierce Chemicals,Rockford, IL). Aseptic techniques were used for cellcultivation, sampling and analysis. Excess-Substrate Induced Uncoupling The effect of excess substrate conditions on biomass yieldwas simulated by varying the initial amount of pyruvate ( S 0 )available for growth, while keeping the initial cellconcentration (  X  0 ) constant. Cells were grown in minimalmedia containing 5, 20, 50 and 100 mM pyruvate in 500 mLvolume Kimax  1 glass bottles. The experiment was carriedout in duplicates and samples were collected at 0, 24, 36, and48 h. The results obtained for biomass growth and substrateutilization (determined by HPLC) were used to model thekinetics of   S. oneidensis  MR-1. Specifically, the number yieldand protein yield, obtained at the end of the experiment(48 h of growth period) were modeled as a function of relative substrate concentration ( S 0 /  X  0 ). Preliminary Uncoupling Experiments Preliminary experiments were conducted to determine anoptimal time for the addition of uncoupler. One parts permillion of 3, 3 0 , 4 0 , 5 tetrachlorosalicylanilide (TCS, AcrosOrganics 1 ) was added to cultures at 0, 12, 24, and 36 h aftermediainoculation to different bottles containing cells grownin 20 mM pyruvate minimal media. Biomass growth andsubstrate removal efficiency were determined for eachtreatmentand compared withthe valuefor acontrol culture.Direct cell enumeration was carried out using a compoundmicroscope and Petroff Hausser counting chamber (Haus-ser Scientific, Horsham, PA).Another experiment was conducted to check the effective-ness of TCS in reducing the biomass of   S. oneidensis  MR-1.For this purpose, 10 ppm of TCS was added to cells grown in10% TSB media, 10 h after media inoculation. Direct cellcount was carried out using a Petroff Hausser countingchamber and the results were compared to a control culture. Uncoupling Via Chemical Addition Three different TCS doses were employed to model theuncoupling effect of this organic protonophore on cellularmetabolism. Specifically 0.5, 1, and 2 ppm of TCS was addedto cells grown in a minimal media containing 20 mMpyruvate, 24 h after media inoculation. The experiment wasconducted in duplicates. Control cultures, without uncou-pler addition, were also included to act as indicators of uncoupling due to excess substrate conditions alone, as20 ppm pyruvate culture was already found to be uncoupledduring ‘‘excess substrate induced uncoupling’’ experiment.While the cultures with TCS addition acted as representa-tives of combined uncoupling actions of excess substrate aswell as uncoupler addition. Samples were taken at 0, 24, 36,and 48 h after inoculation of the growth media. Biomass yield was measured in the same fashion as in the caseof excess substrate induced uncoupling experiment. Theobserved number yield was modeled as a function of relative uncoupler concentration ( C  u /  X  ). Error Analysis In order to account for the errors in the quantification of various parameters, repeated measurements of the samesamples were carriedout so that 95%confidence levels couldbe determined (Taylor, 1982). These confidence levelscorrespond to an uncertainty of 11.7% in determining boththe biomass yield and  S 0 /  X  0 , when cell count was used as themeasure of biomass. A corresponding uncertainty level of 9.3% was observed for the case where protein levels wereused to quantify the biomass. Since the uncouplingcoefficient ( E  u ) is dependent on the observed yield ( Y  obs ),the same uncertainty levels were used for plotting it. Model Development A number of models have been proposed to describe thekinetics of uncoupled cellular metabolism. The earliestmodel assumed a constant maintenance energy requirementto account for all the energy not used for biomass synthesis(Pirt, 1965). It related the biomass yield ( Y  ) to maintenancecoefficient ( m ), specific growth rate ( m ), and true ortheoretically maximum yield ( Y  G ) as1 Y   ¼ m m þ 1 Y  G (1)This model works well for substrate-limited cultureswhere there is little carbon and energy to be wasted.However, it was later observed that the yield ( Y  ) and specificgrowth rate ( m ) are not necessarily directly proportional toone another under common growth conditions because of partial dissociation between catabolism and anabolism dueto excess substrate conditions. Under such conditions, therate of anabolism is reduced due to uncoupling whereasthe rate of catabolism remains unchanged; this causes areduction in the observed biomass yield. Several modelshave been developed to explain this phenomenon (Pirt,1982; Tsai and Lee, 1990; Zeng and Decker, 1995). Liu (1996) proposed a model to describe the relationshipbetween observed biomass yield ( Y  obs ) and the ratio of the 1354  Biotechnology and Bioengineering, Vol. 99, No. 6, April 15, 2008  initial substrate concentration ( S 0 ) to initial biomassconcentration (  X  0 ) as follows1 Y  obs ¼  1 ð Y  obs Þ max þ  1 ð Y  w Þ min  ð S 0 =  X  0 Þð S 0 =  X  0 Þþ K  s = x (2)Here  Y  obs  is the observed growth yield, ( Y  obs ) max   is theobserved growth yield under substrate-limited conditions,( Y  w  ) min  is the minimal energy spilling related growth yield,and  K  s/x   is a saturation constant related to  S 0 /  X  0  ratio(Liu, 1996). ( Y  obs ) max   is same as the parameter ‘‘ Y  ’’ in Pirt’smodel (Eq.1)and includesthetheoreticalmaximumgrowth yield as well as the maintenance requirements. ( Y  w  ) min represents the minimal yield wastage due to energy spilling;while  K  s/x   is a Monod-like saturation constant relating  S 0 /  X  0 to substrate wastage rate (Liu, 1996; Zeng and Decker,1995). This model works well for substrate-sufficientcultures, where growth is limited by something other thanthe growth substrate. Under substrate-limited conditions(growth limited only by substrate availability), the observedgrowth yield ( Y  obs ) approaches the theoretical maxi-mum observed yield ( Y  obs ) max  . The constant ( Y  obs ) max   isdetermined graphically as the intercept of curve drawnbetween (1/ Y  obs ) and  S 0 /  X  0 . Similarly, a graph between1/[(1/ Y  obs )  (1/( Y  obs ) max  )] and 1/( S 0 /  X  0 ) would yield( Y  w  ) min  as the slope, and the product ( K  s/x  )( Y  w  ) min  as theintercept. In this way all the three constants, ( Y  obs ) max  ,( Y  w  ) min , and  K  s/x   can be determined (Liu, 1996).A similar model has been proposed to describe the effectof chemical uncoupler addition on the biomass yield asshown in Equation (3). Here,  C  u  is the initial uncouplerconcentration, and  K  u/x   is a saturation constant related to C  u /  X  0  (Liu, 2000).1 Y  obs ¼  1 ð Y  obs Þ max þ  1 ð Y  w Þ min   C  u =  X  0 C  u =  X  0 þ K  u = x (3)None of the existing models describe the effect of simultaneous presence of excess substrate and chemicaluncouplers; although it seems likely that addition of chemical uncouplers could augment the effect of excesssubstrate conditions. We propose the following empiricalexpression to model the effect of both excess substrate andchemical addition simultaneously.1 Y  obs ¼  1 ð Y  obs Þ max þ  1 ð Y  w Þ min   S 0 =  X  0 S 0 =  X  0 þ K  s = x þ  1 ð Y  wu Þ min   C  u =  X C  u =  X  þ K  u = x (4)In this equation, ( Y  wu ) min  is the minimal energy spillingrelated growth yield under uncoupler addition conditionsand  X   is the biomass concentration at the instant of uncoupleraddition.( Y  wu ) min  and K  u/x  are similar to ( Y  w  ) min and  K  s/x   respectively, and reflect the effect of energy-spillingdue to uncoupler addition. All other parameters are asalready defined for Eqs. (2) and (3). In absence of a chemicaluncoupler, Eq. (4) reduces to Eq. (2). Under substrate-limited conditions, the observed yield ( Y  obs ) approaches thetheoretical maximum value, ( Y  obs ) max  .To determine the values of the constants in Eq. (4), cellswere grown in media having different values of ( S 0 /  X  0 )without addition of uncoupler. This reduces the governingmodel (Eq. 4) to Equation (2) and the parameters ( Y  obs ) max  ,( Y  w  ) min  and  K  s/x   can be determined as previously explained.Using these parameters, the first two terms on the right-hand side of Equation (4) are known for given values of ( S 0 /  X  0 ). The other unknowns (( Y  wu ) min  and  K  u/x  ) can beobtained by varying the values of   C  u /  X   in cultures withknown values of   S 0 /  X  0 ; as already described. Once theseparameters are known, Equation (4) can be used to predictthe values of observed yield ( Y  obs ), based on just ( S 0 /  X  0 )and/or ( C  u /  X  ) (if uncoupler was also used).The degree of uncoupling is computed by a parametertermed as uncoupling coefficient ( E  u ) and is defined as thechange in biomass yield due to uncoupling, as a fraction of maximum possible yield (Liu and Chen, 1997; Liu et al.,1998). Mathematically, it can be expressed as E  u  ¼ð Y  obs Þ max  Y  obs ð Y  obs Þ max (5)This parameter quantifies the fraction of substrate that iswasted as a result of metabolic uncoupling. The uncouplingcoefficient is calculated by using the observed yield ( Y  obs )obtained from the experimental results as well as ( Y  obs ) max  ,determined as described earlier. This parameter can also bepredicted by using the simulated values of   Y  obs , determinedfrom Equation (4) as a function of ( S 0 /  X  0 ) and ( C  u /  X  ), withconstant value of the parameter ( Y  obs ) max  , already calculatedfrom experimental data (Table I). It can be effectively utilized to distinguish between the amount of uncouplinginduced by excess substrate and chemical uncoupleraddition. Table I.  A summary of the modeling parameters, based on number yield,used in the study.Parameter Value Units( Y  obs ) max   2.5  10 8 (cells/mL)/mM pyruvate( Y  w  ) min  3  10 7 (cells/mL)/mM pyruvate K  s/x   4.06  10  5 mM pyruvate/(cells/mL)( Y  wu ) min  5  10 7 (cells/mL)/mM pyruvate K  u/x   5.09  10  9 ppm TCS/(cells/mL) Saini and Wood: Metabolic Uncoupling of   Shewanella  1355 Biotechnology and Bioengineering  Results Excess-Substrate Induced Uncoupling The biomass yields observed in the batch experiment with 5,20, 50, and 100 mM pyruvate media have been plotted asa function of relative substrate concentration ( S 0 /  X  0 ).Figures 1 and 2 illustrate the variations in observed number yield and protein yield, respectively. The observed data isshown by solid points, model fit by continuous lines; whilethe error bars represent 95% confidence levels. The observedand model-simulated values of the uncoupling coefficient( E  u ), obtained by using number yield data (Fig. 3) are shownby solid points and continuous lines,respectively. Thevaluesof various modeling parameters used in Equation (4), aresummarized in Table I.Figure 4 illustrates the average protein content (protein/cell) of the cells at the end of the experiment ( t  ¼ 48 h). Theaverage cell protein content has been determined as a ratioof total protein content (ppm protein) to the cellconcentration (cells/mL). Preliminary Uncoupling Experiments The effectiveness of TCS was tested by examining theresponse of cell growth to addition of the uncoupler. Theexperimental results for changes in cell count, as comparedto a control culture are illustrated in Figure 5. Theexperiment conducted to determine the optimum uncou-pler addition time revealed that TCS addition at 24 hresulted in good cell growth ( > 5  10 8 cells/mL) and a highoverall substrate removal efficiency (  97%) in 48 h of experimentation. At other dosing times, either the cellconcentration or the substrate removal efficiency were toolow to be practical (0 and 12 h dosing times); or theuncoupler had negligible effect on biomass growth (36 hdosing time) (data not shown). Uncoupling Via Chemical Addition Figures 6 and 7 illustrate the number yield and protein yieldrespectively, upon addition of different TCS doses, as afunction of relative uncoupler concentration ( C  u /  X  ). Themodel predictions generated by fitting Equation (4) to theobserved number yield data are shown by solid lines (Fig. 7).The values of constants, ( Y  obs ) max  , ( Y  w  ) min , and  K  s/x  previously obtained in the excess substrate induceduncoupling experiment, were used in the current experi-ment.Thecurrentdata set was usedto computethe valuesof ( Y  wu ) min  and K  u/x  , as previously described. All the modelingparameters used in the study are summarized in Table I. Figure 1.  Effect of relative substrate concentration ( S  0  / X  0 ) on number yield. Figure 2.  Effect of relative substrate concentration ( S  0  / X  0 ) on protein yield. Figure 3.  Uncoupling coefficient in excess-substrate cultures, using numberyield. 1356  Biotechnology and Bioengineering, Vol. 99, No. 6, April 15, 2008
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