ELSEVIER

Journal of Microbiological Methods 27 (1996) 183-197

Journal Microbiological Methods

Community analysis by Biolog: curve integration for statistical analysis of activated sludge microbial habitats
James B. Guckerta*, Gregory J. Carrb, Troy D. Johnsonb, Burt G. Hamm, Daniel H. Davidsona, Yoshiharu Kumagai
Environmental Science Department, Ivorydale Technical Center, The Procter and Gamble Company, Cirtcinnati, OH 45217- 1087, USA bBiostatistics and Medical Surveillance Department, Miami Valley Laboratories. The Procter and Gamble Company, Cincinnati, OH 45253-8707, USA Professional and Regulatory Services-Asia, Kobe Technical Center, The Procter and Gamble Company, Kobe. Japan

Received 19 August 1996; revised 19 September 1996; accepted 19 September 1996

Abstract
Biolog MicroPlates are 96-well plates that contain pre-dried carbon sources and a tetrazolium violet redox dye that turns purple if added microorganisms utilize the nutrients. We have measured absorbance changes due to tetrazolium dye response for microbial communities found in activated sludge and laboratory models of these wastewater communities. To analyze the absorbance versus time data, we export and organize the data into spreadsheets and use a trapezoidal approximation to determine the area under the absorbance versus time curve for each well. The Excel-based trapezoidal approximation has been shown to be in agreement with a more complex curve fitting routine that used SAS to fit a log-logistic function to each curve, and then determined the area under each curve. This data analysis procedure has the advantage of collapsing the absorbance versus time curves down to a single value that integrates information from the entire incubation period. This single value incorporates the lag phase, the rate of development and the extent of dye development for each well. The area under the curve is then used for statistical testing to compare individual carbon source utilization, or in a multivariate pattern analysis of community metabolism of the Biolog MicroPlate carbon sources. Examples of the use of this analysis are given for microbial communities of activated sludge and laboratory models of activated sludge maintained with different types of feedstocks.
Keywords:

Industry;

Activated sludge; Biodegradation Statistics; Wastewater treatment

testing;

Biolog;

Microbial

ecology;

Ministry

of International

Trade and

1. Introduction Biolog MicroPlates are 96-well plates that contain pre-dried carbon sources and a tetrazolium violet redox dye that turns purple if the added microorganisms utilize the carbon source [l]. When iso*Corresponding author. Tel: + 1 513 6263373; fax: + 1 513 6263522: email: guckert.jb@pg.com.

lated strains are evaluated, the pattern of responses over a Biolog plate can be compared to databases to establish probable identifications [2]. There are severa1 different arrays of carbon sources designed to optimally identify Gram-negative (GN Biolog plates), Gram-positive isolates (GP Biolog plates) or yeasts (YT Biolog plates). In addition, empty plates (MT Biolog plates) are available that contain no carbon sources, but do contain the tetrazolium dye.

0167-7012/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PIZ SO167-7012(96)00948-7

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Biolog plates were originally designed to be evaluated after a 4 or 24 h incubation and compared with established databases of pure cultures [3,4]. Recently, microbial ecologists have been using Biolog plates to investigate carbon-source utilization patterns for mixed microbial communities. This technique has been used with soil microbial communities [5-S], aquatic samples [5], [D.M. Lee and J.B. Guckert, unpublished data] and wastewater treatment communities [9], [and this report]. In addition, the MT Biolog plates [lo], which contain no added carbon sources, have been used to screen bacterial strains for their ability to biodegrade toxic organics [l l] and surfactants [12]. Community-level Biolog analysis is accomplished in four steps: (1) process the sample to create a suspension of microorganisms, (2) inoculate the Biolog plate(s) with aliquots of the suspensions, (3) incubate the plate while monitoring color development in each well, and (4) analyze the results. Victorio et al. [9] has published a comparison of sample processing techniques for wastewater activated sludge and has concluded that homogenization produced the most representative community with the best recovery. Inoculum cell density has been shown to influence color development in Biolog plates [5,6,8]. A minimum number of metabolically active cells (about lO*/ml based on [8]) are required to produce an observable color change. This has resulted in the recommendation (e.g. [S]) that all microbial suspensions be adjusted to a standardized cell density prior to inoculation into Biolog plates. This is the approach suggested by Biolog, Inc. [3,4] when the plates are used to identify pure cultures. An alternative that has been proposed [5] is to adapt the data analysis to account for different inoculum densities. A standardized reference point, the Average Well Color Development (AWCD), is calculated as the mean of the absorbance values for all 95 response wells per reading time [5]. All well responses are then normalized to the AWCD for each plate to account for different inoculum densities [6]. Although color development in Biolog plates is often monitored many times over the course of an incubation period, data analysis has been generally limited to a few selected time points for microbial community analyses [5-91. The absorbance versus incubation time curves contain additional informa-

tion not available from any single time point analysis; such as, lag times, rates of color development and maximum absorbance. Several researchers have discussed the probable utility of an analysis that could take into consideration this additional information [6,8]. In this report, we describe a new approach to Biolog data analysis that incorporates all of the additional information from the absorbance versus incubation time curves into a single number using a PC-based curve integration analysis. Data sets for several microbial communities (described below) based on this new approach are then analyzed using both univariate and multivariate analyses. On the basis of our results, the use of curve integration provides a powerful tool for the analysis of microbial community metabolic patterns. Several researchers have discussed limitations of the Biolog method, especially related to the differential growth of microorganisms that occurs in the individual Biolog wells [6-81. While we acknowledge that changes in community structure can occur during Biolog incubations, we interpret all Biolog results as a function of the original microbial community structure from the sample. The rate and extent of utilization of any particular carbon source on a Biolog plate will be related to the original microbial community structure and metabolic capacity for that sample. Since the community will have a single carbon source to utilize in each well of the Biolog plate, the microbial community will likely change independently in each of these wells. However, the measured Biolog response is still related to the functional potential of the original community [5]. Therefore, we believe that a comparison of microbial communities based on the 95-carbon source array available from a Biolog plate is an appropriate relative measure of the metabolic diversity of these communities. The results discussed in this report indicate significant shifts in carbon source utilization patterns that are relevant to the research questions being addressed for microbial communities of fresh activated sludge as compared to laboratory models of these communities, as described below. Wastewater treatment plants (WWTPs) are important microbial habitats that remove wastes by a combination of biological and physical processes. To facilitate biological removal, WWTPs provide a managed habitat for a complex microbial community

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18.5

that is maintained to breakdown biodegradable wastes [13]. Although this biodegradation of wastes is of great importance to human society and receiving stream water quality, there has been minimal research into the microbial ecology of these habitats [9]. Recently, the microbial ecology of these communities has been evaluated with such techniques as gene probes [14], enzyme activity profiles [15], and Biolog [9]. For the purpose of regulatory clearance of new chemicals, such as consumer products ingredients, sludge from a WWTP is used to evaluate the biodegradability of these new chemicals. It is normally stipulated that the WWTP should treat predominantly domestic sewage, and be free from major specific pollution. The sludge used in the Organization for Economic Cooperation and Development (OECD)-Japanese Ministry of International Trade and Industry (MITI) biodegradation test (3OlC), however, is different from other OECD readybiodegradation tests [16]. This test requires that the WWTP sludge microbial community initially collected be maintained in a Semi-Continuous Activated Sludge (SCAS) unit [17] on a glucose and peptone feed for at least I. month before biodegradation testing can begin. A.s part of a global, collaborative program evaluating an array of methods for the community-level changes that occur during the MIT1 cultivation process, 13iolog was selected as a measure of changes in metabolic diversity. The results shown in this report highlight our new Biolog data analysis procedure, using the comparisons of carbon-source utilization profiles for fresh WWTP sludge with SCAS unit communities maintained on fresh sewage or synthetic feeds (such as the MIT1 glucose and peptone).

directly to Procter and Gamble Laboratories for analysis. The Middle East Fork WWTP is a 7 million gallons a day secondary treatment plant. Middle East Fork serves a residential population of over 10 000 in Clermont County, OH, with about 15% of its flow from industries. The Polk Run WWTP is a secondary treatment plant with a capacity of about 6 million gallons a day. Polk Run services a rapidly growing area of northern Cincinnati (Hamilton and Warren counties) with about 5% of its influent coming from industry. The Sycamore WWTP is a secondary treatment plant with a capacity of about 6 million gallons a day. Sycamore serves a population of over 30 000 in Hamilton County, with industrial contributions accounting for about 1% of its influent [ 181. Sufficient activated sludge was collected to allow 1.5 1 of sludge for each laboratory unit (described below) to be set up. Sludge was transported in a container with ample headspace to maintain aerobic conditions in the sludge. When possible, aeration was maintained during transportation or restored at the earliest opportunity. In all cases, sludge was set up with aeration in the laboratory units within 24 h of collection.

2.2. Semi-Continuous unit set-up

Activated

Sludge (SCAS)

2. Materials

and methods

2.1. Waste Water T,reatment Plants (WWTPs) sampled Three activated sludge WWTPs in the Greater Cincinnati area were sampled for activated sludge. These WWTPs discharge into the watershed of the Little Miami River, OH, USA 1181. In each case a volume of sludge was collected and transported

SCAS units are used as batch models of WWTP microbial communities to study WWTP processes, chemical fate and biodegradation potential [ 171. SCAS units used in this study consisted of Plexiglas cylinders with a coned bottom to prevent settling of sludge solids during aeration. The cylinders were approximately 60 cm tall with an inside diameter of 8 cm. An air dispersion tube introduced air at a rate of 85 -+5 ml/min. The air was introduced at the lowest possible point in the cone to maximize the dispersion of air bubbles through the sludge (this is accomplished by aiming the tube down at a 45 angle toward the bottom of the cone). Another tube, used to drain and/or sample the unit, enters the unit horizontally at the 500 ml mark. This tube was kept clamped shut during normal operation to prevent loss of mixed liquor suspended solids. On 20 April 1995, each SCAS unit received 1500 ml Polk Run WWTP activated sludge at a total suspended solids level of

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2000-3000 mg/l. SCAS units were maintained 23.5 h aeration schedule. 2.3. SCAS unit feeding

Once a day, the air to the SCAS units was shut off and the solids were allowed to settle for 30 min. The drain line was then opened and the settled supernatant allowed to drain from the unit down to the 500 ml mark. The drain line was then clamped off, and the units fed. Three different feeds were used. They were: (1) MIT1 Feed (2) Ludzack feed, and (3) Sewage feed. MIT1 Feed is based on the prescribed feed required by the MIT1 Biodegradation Test [16]. This synthetic feed consists of a stock solution of 1 g each of peptones, glucose and KH,PO, dissolved in 2 1 of deionized water. After settling and draining of the supernatant, 1000 ml of the stock solution was added to the unit to bring the final volume to 1500 ml and aeration resumed. Ludzack is another formulation of synthetic sewage ( [19] as cited in [20]). The stock solution consisted of 30 g n-glucose, 20 g nutrient broth, and 13 g K,HPO, dissolved in a final volume of 1 1 of deionized water. After settling and draining of the supernatant, the SCAS unit is fed 20 ml of this stock solution plus 980 ml tap water to bring the final volume to 1500 ml and aeration resumed. Sewage Feed was unmodified sewage obtained once a week from the gravitational thickener of the Polk Run WWTP. The gravitational thickener is just downstream of the activated sludge basins and provides a higher concentration of organics to match the Chemical Oxygen Demand loading of the synthetic feeds (described above) used. After settling and draining, the unit was fed 1000 ml of Polk Run WWTP sewage to bring the final volume to 1500 ml and aeration resumed. All stock solutions and fresh sewage were stored at 4C and warmed prior to use. 2.4. Biolog analysis Biolog is a 96-well MicroPlate technique originally designed to use metabolic patterns to identify pure cultures of bacteria [l]. In this study, Biolog GN (Gram-negative), GP (Gram-positive), and MT (empty) MicroPlates were used to evaluate community-level metabolic responses. GN [4] and GP

[3] Biolog plates contain different arrays of carbon sources along with different inorganic nutrients. MT plates [lo] do not contain any carbon sources, and have been used as a negative control to ensure that responses noted for GN and GP plates are not due to carry-over of carbon sources from the activated sludge matrix. SCAS units were sampled on 8 August 1995, 110 days after their set-up. Three separate samples were collected and analyzed from each SCAS unit. While the units were aerating, a 20-50 ml aliquot of sludge was drained from each SCAS unit. Grab samples of activated sludge were collected on 18 December 1995 from the Polk Run, Middle East Fork and Sycamore WWTPs. Three separate samples were collected and analyzed from each WWTP. Sludge samples from SCAS units and WWTPs required additional preparation steps before microbial communities could be inoculated into Biolog MicroPlates. Microorganisms are removed from the sludge solids by homogenization using a Waring Commercial Laboratory Blender (model 34BL97, 1 min at speed setting #2 at room temperature) with a 355 ml stainless steel attachment and stainless steel blades and lid (Eberbach Corporation). Sequential extractions by homogenization have recovered microbial communities with similar Biolog profiles, suggesting that each extraction removes a representative portion of the intact sludge microbial community [9]. Homogenized sludge samples were placed into 50 ml glass, screw-cap, centrifuge tubes. Suspended microorganisms were then separated from the other material by centrifugation using a Clay Adams Dynac benchtop clinical centrifuge (7OOXg for 5 min at room temperature). The resulting supernatant was fairly turbid. The supernatant was removed by decanting, or by pipette from below the surface if surface grease or oil was observed. Following the established Biolog procedures [3,4,10], the supernatant was diluted with phosphate buffer as necessary to get the turbidity to within a range of 0.25 to 0.35 absorbance units at 420 nm using a Hewlett Packard 8452A Diode Array Spectrophotometer. The stock solution of phosphate buffer was 12.36 g Na,HPO,, 1.80 g H,PO, and 85.0 g NaCl in 1 1 deionized water, filtered (0.2 ,um) and stored at 4C. The working solution for dilutions used 100 ml of this stock in 1 1 of deionized water.

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The suspension was then poured into a reagent reservoir and 150 b~l aliquots were added to each well of the Biolog MicroPlates using a multi-channel pipettor (Brinkman Transferpipette-12). Typically, one Biolog GN, GP and MT MicroPlate were set up for each replicate sample. Biolog plates were evaluated for absorbance changes at 590 nm using a Molecular Devices !$PECTRAmax Model 250. Following an initial (time 0) reading, Biolog plates were incubated at 25C. The Biolog plates were read for absorbance at 590 mn since respiration of the carbon source in each well will cause the tetrazolium dye to turn purple and abssorb light at 590 nm. Plates are read periodically (usually 9-12 times) over a 3 day period. For example, plates were commonly read at 0, 17, 21, 25, 41: 45, 49, 65 and 69 h. One representative plate could also be read continuously (every 30 min) over the entire incubation time, if desired. This kinetic-style analysis could be extended to all samples with the use of an automated plate stacker/reader. 2.5. Statistical
analysis

constant variance. PCA was then used to evaluate separations of samples based on the 95 carbon source utilization pattern. Scores for variable loadings were evaluated to determine which carbon sources provided the largest discrimination power, and these individual carbon sources were evaluated individually. For the purposes of this report, no hypothesis testing (e.g. ANOVA) was conducted due to the level of replication available in this example data set. Individual SCAS unit feeds were not replicated in separate SCAS units, so replicates show levels of variability from pseudo-replicates rather than true treatment effects [22].

3. Results and discussion 3.1. Biolog absorbance

curves

Results were automatically saved into data files using Soft MAX Pro (version 1.2.0). Separate data files were created for each of the 9-12 readings conducted on each 13iolog plate over the incubation time. The data from these individual readings was complied into a single Excel spreadsheet for each plate. An automated method to compile these files also provided a calc:ulation of area under the curve for each well of the plates. Details of this analysis procedure are discussed in Section 3 and Appendix A sections. Results Ibr analysis are expressed as net area under the curve where the area under the curve for well Al (water control) is subtracted from each of the other 95 wells for a plate. In the development of this method, data files were first evaluated using a curve-fit routine available in SAS [21], as discussed further in Section 3. Biolog results were first plotted by well number to visualize trends. Profiles of Biolog results were also analyzed by multivariate analysis with the pattern recognition software package Ein*Sight (version 2.5, Infometrix, Inc.). Principal component analysis (PCA) was conducted on the net area under the curve data by first autoscaling the data to a mean of 0 and

Analysis of microbial communities by Biolog produces a very large data set. Each of the 96-wells per MicroPlate has 9- 12 absorbance measurements taken over the 3-day incubation time. Therefore, there are 96 curves that need to be analyzed. Fig. 1 uses selected data from one of the MITI-fed SCAS units to show how results from 24,48 and 65 h could be very different for the same Biolog plate. Absorbance versus incubation time curves can be used to develop summary information about the well color development in each well. The rate of increase in absorbance might be considered, though it would not account for a lag phase. For example, 2-aminoethanol (H7) and Tween 40 (A5) appear to have similar rates of increase, but because of differences in the length of the lag phase, the overall utilization of these carbon sources is very different (Fig. 1). Also, a maximum absorbance could be evaluated, though this will not indicate the rate of increase or duration of color development. For instance, 2aminoethanol (H7) finishes the incubation at a higher absorbance value than glycerol (H9), but evaluating the entire curve clearly shows that glycerol was more rapidly and extensively used over the entire incubation time (Fig. 1). Garland and Mills [5] introduced the concept of Average Well Color Development (AWCD) for community-level Biolog data analysis. A plot of AWCD over time (Fig. 2) for this data set is similar

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2.0

A5 /,/ MAX. OD / //
1)

E c 1.5

z s J 1.0 El P $ 0.5 9 0.0

I I I i I I I

LAG
I I I ! / I I

10

20

30

40

50

60

70

80

Incubation time in Hours 10 20 30 40 50 60 70 80

Incubation time in Hours

Fig. 1. Optical Density versus time curves for four wells of Biolog GN MicroPlate inoculated with a MITI-fed SCAS unit microbial community. Optical density is expressed as absorbance units at 590 nm. The carbon sources are labeled by well number. Al, water (control); A5, Tween 40; H7, 2-aminoethanol; H9, glycerol. These carbon sources were selected to show that picking any one time point to evaluate the Biolog plate results can give different results for the same plate. At 24 h, A5>H9>AI >H7. At 48 h, A5>H9>H7>Al. At 65 h, A5>H7>H9>>Al. Also, the three key factors that make up the optical density versus time curves are marked, the lag phase, the maximum rate of increase and the maximum optical density. The analysis for any one curve needs to take into consideration all of these factors.

Fig. 2. Average Well Color Development (AWCD) is the mean of the blanked (At subtracted) absorbance values for all 95 response wells per reading time (see [4] for details). This AWCD versus time curve is for a GN plate inoculated with a MITI-fed SCAS unit microbial community. This curve shows that this active community had, on average, very little lag time in the development of dye within the Biolog plate wells. Development was consistent and linear over the entire 3-day incubation period. The number of positive wells (defined as blanked absorbance values >0.25) over time increased rapidly for the first 24 h. At this time, the number declined slightly, possibly due to an increase in the control well (Al) absorbance value (Fig. 1). The number of positive wells then increased until about 50 h of incubation when it began to plateau.

to one shown by Garland (see Fig. 1 in [6]), although it appears that activated sludge communities have a much shorter lag time than the soil communities evaluated by Garland [6]. AWCD development was consistent and linear over the entire 3-day incubation period. The total number of positive wells, described as having a net (Al subtracted) value BO.25 absorbance units, is also shown in Fig. 2, and is again similar to the results shown in [6]. The number of positive wells over time increased rapidly for the first 24 h. After 24 h, the number declined slightly, possibly due to an increase in the control well (Al) absorbance value (see Fig. 1). The number of positive wells then increased until about 50 h of incubation when it began to plateau. These results indicate that, at the end of a 3-day incubation period,

no new carbon sources were being utilized, but that, on average, the microbial community activity continued to consume the carbon sources and increase color development of the Biolog dye. The AWCD method for Biolog analysis was developed to account for differences in inocula densities. However, this method still requires data analysis at various incubation times. Garland [6] proposes to define the analysis point based on the time required to achieve a certain AWCD absorbance value, such as 0.75. Garland [6] also showed how multivariate classifications could be influenced by differences in AWCD set points (e.g., 0.25, 0.5, 0.75, 1.00). Our integrative approach (described below) requires an inoculum density adjustment, but provides a method of analysis more independent of incubation time.

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3.2. Net area under the curve approach Our approach to 13iolog data analysis has been to calculate area under the absorbance versus time curve. We feel that this number best summarizes the color development information because its value will reflect differences in lag phases, rates of increase, maximum optical densities, etc. We initially used a kinetic Biolog data set in which we obtained 98 data points collected at approximately every 30 min over a 3-day period. The 98 data points for each of the 96 wells was used as input for a curve-fitting routine in SAS [21]. SAS was successful at fitting log-logistic curves to each of the 96 sets of data. We then used the same data set and truncated all but 12 of these observations. These 12 observations were selected so they reflected actual times we would plan to observe the Biolog plates over a 3-day incubation period. No significant differences were found between the curves, indicating that we could simplify the procedure by taking fewer data points to obtain the same result (G. Carr, unpublished data). Even with the fewer data points per Biolog plate, the SAS curve-fitting and area integration routine was very time consuming in both real time and computer CPU time. We then explored an additional simplification in which an estimate of area under the curve was developed by summing the trapezoids formed when drop lnes were drawn from each data point to the X axis. Details of the calculation used for this approximation are provided in the Appendix A. A comparison for all of the SCAS Biolog results (discussed below) was made. Area was calculated for all 1728 curves (96 wells on each of 18 MicroPlates) using the SAS curve-fitting routine along with an area integration as compared to an Excel-based calculation of the ttapezoidal approximation to area under the curve, shown in the Appendix A. Area values greater than 1 unit by both methods were then analyzed. A brief sample of results is shown in Table 1, with summary stiatistics for the 1608 wells with area >l unit. The average absolute value difference in area under the curve was 0.80 units, with a maximum difference of 7.3 1 (Table 1). The average percent difference between the two approaches was 3.19%. The summary of data (Table 1) shows that the average area under the curve was about 41 area units for both, so an average difference of 0.80 units

is minor (<2%). These results indicate that the trapezoidal area under the curve is a reasonable estimation of the area under the Biolog absorbance versus time curves, and thus was used for all further Biolog evaluations. 3.3. Biolog resu.lts for laboratory SCAS units

Large tables of data were obtained for the net area under the curve (with well Al subtracted) for the SCAS units and the Ohio WWTPs. As a first step to analyze these complex data sets, profile plots were made of the net area results by well number, which can be related to carbon source using the crossreferences provided in Biolog literature [3,4]. When the GN results for the MITI-fed SCAS unit community were evaluated (Fig. 3), it was apparent that while many carbon sources were utilized by the indigenous microbial communities (high net areas), there was also a significant number that were not. 3.4. Results from Biolog MT MicroPlates The MT plate results were also plotted (Fig. 3) to evaluate what should be a negative control; only nutrients and dye, no carbon sources. The MT results for the SCAS units indicated a cycle of positive values across the plate (Fig. 3). The highest values in these cycles were found in the middle of the 96-well plate, while the lowest values were located at the edges. A communication with technical experts at Biolog, Inc. suggested that we may be seeing an evaporation phenomenon. Keeping the Biolog incubator hydrated with a pan of water decreased the amplitude of the MT results, but cycles due to unknown phenomena were still present. To put the values of the MT results into perspective, we routinely plot the GN or GP values with the MT values on the same graph, as shown for the MITI-fed SCAS GN results (Fig. 3). Due to space constraints only the MITI-fed SCAS GN data, which had the largest area values for an MT response, are shown here (Fig. 3). The Sewage SCAS unit had a low and uniform MT result, in comparison with the GN or GP Biolog plates. The Ludzack SCAS unit had a similar response, with a low and uniform MT result. The MITI-fed SCAS unit had the largest MT plate response (Fig. 3). However, our evaluation of the

190 Table 1 Comparison Well

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of integration

methods for Biolog data sets Area under SAS best-fit curve 89.2465 68.4507 68.4111 72.7837 19.5848 81.0536 80.6474 84.3316 81.2219 58.7679 72.0657 80.9 74.7898 63.9544 88.8478 97.2798 27.1123 12.8642 58.1416 33.7667 18.7633 73.1211 55.53 16 48.0974 55.0856 56.9821 45.2828 60.5233 1.516 59.0321 53.7607 85.3295 50.5604 7.1777 53.103 39.4426 Trapezoidal estimate of area under the curve 91.951 73.3128 69.2353 70.4263 18.4186 79.3904 85.301 85.4424 79.155 60.3558 67.2839 75.2803 71.264 59.3226 90.2037 97.6841 22.4638 9.0734 57.6789 33.9934 15.9254 71.5239 49.8672 46.1945 48.593 55.0411 42.5561 63.0733 3.1708 57.0506 52.0241 88.7305 47.3626 3.5246 50.2051 38.7184 40.59 20.17 98.78 1.06 41.57 1608 Absolute value difference 2.70 4.86 0.82 2.36 1.17 1.66 4.65 1.11 2.07 1.59 4.78 5.62 3.53 4.63 1.36 0.40 4.65 3.79 0.46 0.23 2.84 1.60 5.66 1.90 6.49 1.94 2.73 2.55 1.65 1.98 1.74 3.40 3.20 3.65 2.90 0.72 0.80 0.85 7.31 0.00 0.57 1608 Percent difference 2.99 6.86 1.20 3.29 6.14 2.07 5.61 1.31 2.58 2.67 6.86 7.20 4.83 7.5 1 1.51 0.41 18.75 34.56 0.80 0.67 16.36 2.21 10.75 4.04 12.52 3.47 6.21 4.13 70.62 3.41 3.28 3.91 6.53 68.27 5.61 1.85 3.19 5.95 70.62 0.00 1.57 1608

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 41 42 43 44 45 46 47 48 49 50 51 52 54 Average S.D. Max Min Median n=

40.73 20.40 98.40 1.07 41.78 1608

Brief data summary showing data from a GN plate inoculated with MITI-fed SCAS community. Summary statistics are for results across GN, GP and MT plates for all SCAS unit communities where area under the curve values were greater than 1 unit,

GN versus MT results (Fig. 3) suggests that even for this worst-case example, the MT results do not significantly overlap with the higher net area responses seen on the GN plates. No further subtrac-

tion of MT results was used for any sample since the response noted in the MT plates does not appear to be due to any carbon source carry-over for these samples. In addition, this subtraction may not be

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191

& d

120

MlTl t 1 SEWAGE LUDZACK t

3 ln
E f

100

60

; O

60

5 I? 3 3 2 Y

40

20

10

20

30

40

50

60

70

60

90 100

Well Number
Fig. 3. Profile plot of Biolog results expressed as net area under the curve (with control well Al subtracted) for each of the 95 response wells (e.g.. well number 2=A2, well number 96=H12) over a 3-day incubation, Results are shown for the MITI-fed SCAS unit microbial community in the Biolog GN (0) and MT (W) MicroPlates. A line is drawn through the data points as an aid in observing the response patterns. Values are given as mean area units irl S.D. for three separate Biolog plates run from three separate samples collected from the same SCAS unit, and then processed individually.

GN

GP

Fig. 4. Results for cr-o-glucose utilization in the Biolog GN (well B6) and GP (well Bl 1) for the three SCAS units maintained with different feeds. Values are mean area units (3-day incubation) 2 1 S.D. for three separate Biolog plates run from three separate samples collected from the same SCAS unit, and then processed individually. In theory, these values between the GN and GP should be identical, and for the Sewage-fed SCAS unit there is good agreement. However, the MITI- and Ludzack-fed SCAS units had lower values for glucose on the GP plate.

warranted to normalize for well-to-well differences as the MT plates are manufactured differently from GN plates. 3.5. Results for Bioiog GP MicroPlates Biolog GP plates were also analyzed for the SCAS units. In Fig. 4, the GN and GP net area under the curve results for glucose are compared for the MITIfed, Sewage-fed and Ludzack-fed SCAS units. The GN results suggest Ithat these SCAS unit microbial communities are about the same in their ability to utilize glucose. Even though the MIT1 and Ludzack communities have bleen cultivated with glucose, it makes some sense that the capacity to utilize this carbon source would be retained in the sewage-fed communities. The results for the GP Biolog plates, however, are very different. These results suggest that the glucose-fed microbial communities of the MIT1 and Ludzack-.fed SCAS units had a lower

capacity to utilize glucose than the sewage-fed communities (Fig. 4). A more likely explanation is that differences between GP and GN plate inorganic nutrients may have influenced this glucose result. Assuming that glucose-fed communities are not likely to lose the capacity to utilize glucose, these results suggest that the MIT1 and Ludzack-fed SCAS communities appear to have been influenced, and likely inhibited, by inorganics found in the GP plates. Our original intent in using both GN and GP Biolog plates was to have an evaluation of 128 unique carbon sources across the two plates, rather than the 95 available from either one separately. In addition, the carbon sources in common between the GN and GP plates were initially thought of as internal replicates within the assay. The glucose results (Fig. 4) discussed above, indicate that the carbon sources contained in both the GN and GP Biolog plates do not necessarily provide this level of replication, In addition, from the analysis below it is also apparent that 95 separate carbon sources provide enough discrimination power for our research ques-

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tions, and analysis of both the GN and GP is unnecessary. Therefore, our future Biolog work will only use GN Biolog plates (with MT plates as controls for comparison but not subtraction) as they appear to provide the most complete and reproducible analysis for these communities. 3.6. Biolog results for WWTPs WWTP samples were analyzed with the GN and MT Biolog plates. The average values for each well on the GN plate are shown for the Polk Run WWTP (Fig. 5). The pattern of carbon source utilization for this fresh WWTP sludge appears to be different from the MITI-fed SCAS unit results shown in Fig. 3. The Polk Run results (Fig. 5) also suggest that Biolog may be a method to quickly differentiate chemical species that are consistently more highly degraded (=large area under the curve) by WWTP communities from those chemical species that are consistently not as well degraded (=small area under the

curve). This information could be particularly useful in the design and development of new, biodegradable, consumer product ingredients. 3.7. Analysis of community-level Biolog results

As an example of how the net area under the curve approach can be used, a comparison is made of Biolog profiles for the three WWTPs and the three different SCAS communities that were maintained with different feeds. Two methods of analysis are presented. One is a multivariate analysis of the entire Biolog profile followed by comparisons of several key individual carbon source responses. The second is a scatterplot of the entire Biolog data set to graphically evaluate trends in the data set. 3.8. Multivariate analysis

g
t; E s ?I

100 90 80

70 60 50 40 30 20
10 0

g ; 3 t 0 5 g
2

h z

10

20

30

40

50

60

70

80

90

100

Well Number
Fig. 5. Profile plots of Biolog results expressed as net area under the curve (with control well Al subtracted) for each of the 95 response wells (well number 2 = A2, well number 96 = H12) over a 3-day incubation. Results are shown for the Polk Run Wastewater Treatment Plant microbial community in the Biolog GN MicroPlates. A line is drawn through the data points as an aid in observing the response patterns. Values are given as mean? 1 S.D. for three separate Biolog plates run from three separate samples collected from the three separate locations within the WWTP, and then processed individually.

When a PCA was conducted for these carbon utilization patterns, the replicate values for each WWTP were very similar (Fig. 6). These first two principal components accounted for 64% of the total variability in the data set. The Middle East Fork and Polk Run WWTPs had similar Biolog metabolic profiles, and these were similar to the profile of the Sewage-fed SCAS unit. The largest separation in this analysis was between the SCAS units fed synthetic feeds (Ludzack and MITI) and the rest of the Sewage-fed SCAS and WWTP microbial communities (Fig. 6). The PCA plot shown in Fig. 6 provides a method to visualize the data and explore its structure, but it is only a first step in the data analysis for these profiles. On the basis of the loadings scores from the PCA, four representative carbon sources (sucrose, L-glutamic acid, xylitol, Tween 40) were selected to more directly compare the SCAS units and the WWTP metabolic patterns. Sucrose appears to be equally utilized by all three WWTPs and the Sewage- and Ludzack-fed SCAS units (Table 2) while the MITI-fed SCAS unit response appears to be enhanced. The MITI-fed SCAS unit also has an enhanced metabolism of the amino acids, such as L-glutamic acid (Table 2). The results with xylitol and Tween 40, however, suggest that the MITI-fed SCAS units lost some key metabolic capacity (Table 2) from the fresh sludge

J.B. Guckert et al. I Journal of Microbiological Methods 27 (1996) 183-197

193

SCAS

al

-15

-10

-5

10

15

20

Principal Component

Fig. 6. Muhivariate Principal Component Analysis for Biolog GN profiles for microbial communities from three WWTPs (V, Middle East Fork; 6, Polk Run; filled hexagon, Sycamore) and three SCAS units maintained with different feeds (0, MITI-feed; A, Ludzack feed; 0, Sewage feed; see text for details). Principal Component 1 (48% of total variability) separates the MITI-fed SCAS unit from the other communities. The addition of the second principal component accounted for 64% of the total variability in the data set and the Ludzack- and MITI-fed SCAS unit communities were clearly separated from the WWTP communities. The SCAS unit that had been fed fresh sewage for 110 days had a metabolic profde more similar to the WWTP microbial communities than the other SCAS units.

microbial community. Xylitol, a hydroxylated pentane derivative (C,II,,O,), had similar Biolog results for the WWTP and the Sewage-fed SCAS unit. The Ludzack and MITI-fed SCAS units, however, had a Biolog result about half of the initial value. The WWTP Biolog response for the non-ionic surfactant Tween 40 is similar to that found in the Sewage-fed SCAS. However, both the Ludzack and MITI-fed SCAS units appear to have much lower values, indicating that these microbial communities have lost some cap,scity to degrade this biodegradable, non-ionic surfactant (Table 2). 3.9. Scatterplot
analysis

Run WWTP (Fig. 7). Results close to the reference line indicate a carbon source response very similar to the Polk Run profile. Results above the reference line indicate that biodegradation activity has been enhanced from the original sludge, results below the line indicate metabolic activity for that carbon source has declined compared to the fresh sludge. When the net areas under the curve for the Middle East Fork and Sycamore WWTPs are evaluated, the results tend to cluster around the reference line (Fig. 7). There does appear to be a trend in which Sycamore WWTP results are below the reference line, but over the range of metabolic activity, the results for all WWTPs appear to be very similar. When the MITI-fed SCAS unit profile is compared to the fresh sludge of Polk Run WWTP, there are many differences (Fig. 7). MITI-fed SCAS profiles suggest an enhanced ability to biodegrade sugars and amino acids. This enhancement is observed for some carbon sources previously at low activity (e.g., area under the curve <20 area units) or much higher activity (area under the curve >50 area units) in the fresh sludge of Polk Run WWTP (Fig. 7). It is likely that this metabolic change is the result of maintenance of the community on the MIT1 feed, which contains sugar and amino acids (in peptone). In addition to an enhancement of sugar and amino acid metabolism, there appears to be a decline in the biodegradation of some more complex carbon sources (Fig. 7). The anionic surfactants (Tween 40 and Tween 80), xylitol and glycogen are several examples of carbon sources that were biodegraded much more readily by the fresh WWTP microbial community. These results indicate that the MITI-feed cultivation process has resulted in a decline in some specific biodegradative capacity.

4. Summary We feel that the analysis of net area under the curve provides a better method for the analysis of Biolog data for microbial community investigations. The area under the curve approach can be automated using PC-based software. The area under the curve measure is sensitive to the differential effects of the lag phase, the rate of increase and the maximum absorbance obtained during the incubation time.

A second approach to community-level Biolog data analysis is graphical using a scatterplot of results compared to {the profile obtained for the Polk

a s
Component Sewage-fed 72.12?0.85 57.61 k3.59 32.6626.93 76.8129.34 SCAS Analysis of WWTP and SCAS unit microbial Ludzack-fed 68.OOk4.67 64.942 1.93 13.20Z2.73 40x9+ 1.33 community SCAS Biolog profiles MITI-fed SCAS 92.70? 14.73 87.08?11.30 18.49?2.02 35.80?4.15 Polk Run WWTP 67.68 -+ 1.65 54.612 1.00 39.36k3.42 67.9552.96 61.6957.70 47.42t2.00 29.27k5.39 60.7526.24 Sycamore WWTP 5 c % z F; 3 P z P. $ from single SCAS units. 2 0s P

Table 2 Examples

of area under the curve results for key carbon sources based on Principal

Carbon source

MEF WWTP

Sucrose (C7 on GN) L-Glutamic Acid (FlO on GN) Xylitol (Cl0 on GN) Tween 40 (A5 on GN)

70.50*3.30 66.27k3.12 39.7323.80 65.3928.06

SCAS unit microbial communities were all originally set up using sludge from Polk Run WWTP. Results are average (2S.D.) area units. Note: Hypothesis testing not conducted since replicates for SCAS units are pseudo-replicates: individual Biolog plates using microbial suspensions

J.B. Guckert et al. I Journal of Microbiological Methods 27 (1996) 183-197

195

L-asparagine 0 L-aspartic acid 6 L-glutamic acid # n\ D-galactose

D-trehalose

sucrose e

0 glycogen

0 Tween 40 ,,.N-acetyl-D-galactosamine l adonitol

20

40

60

80

100

Polk Run WWTP Sludge Biolog Response by Area

Fig. 7. A scatterplot of Eliolog GN responses (average net area under the curve over a 3-day incubation) comparing the Middle East Fork WWTP (A), the Sycamore WWTP (Cl) and the MITI-fed SCAS unit (0) community metabolic profile to that obtained for the Polk Run WWTP (plotted on the Xaxis).Values close to the reference line are in good agreement with the Polk Run values.Values above the reference line have an enhanced aoility to utilize that carbon source, values below have an inhibited ability, as compared to the fresh WWTP sludge from the Polk Run WWTP. The other WWTPs have values very close to the reference line, indicating similar metabolic profiles for all WWTPs. The MU-fed :SCAS community appears to be enhanced in the utilization of many sugars and amino acids and inhibited in the utilization of more complex carbon sources. such as Tween 40 and xylitol.

Since the integration is conducted over the course of the entire incubation time, this data analysis approach is not dependent on set points developed arbitrarily by the researcher. Biolog profiles from WWTP and SCAS unit microbial communities provided several exam-ples of how these data sets can then be analyzed. The MT MicroPlate results from these examples highlight the need to minimize evaporation in the incubating Biolog plates. The comparison of GN and GP results in these examples suggests that inhibitory effects may be complicating the interpretation of results from the GP plates. On the basis of these results, we suggest that the use of

GN Biolog MicroPlates (with MT plates as controls) provides the reproducibility and resolution needed to evaluate changes in metabolic diversity of mixed microbial communities. The preliminary results for the WWTP and SCAS unit comparison also show a practical use of this data analysis technique to address important questions in microbial ecology. In this case, the microbial communities of laboratory SCAS units were maintained with a similar metabolic diversity to the WWTP activated sludge communities when the SCAS units were fed real sewage. When SCAS units were maintained on synthetic feeds of simple carbon sources, the microbial com-

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munitys ability to degrade some simple carbon sources increased, but the ability to utilize more complex carbon sources declined, based on the area under the curve analysis of the community-level Biolog method.

Acknowledgments Comments and suggestions for the Biolog analysis procedure were provided by Dr. L. Forney (Center for Microbial Ecology, Michigan State University), Dr. T. Nishihara (Osaka University, Japan) and Dr. M. Nasu (Osaka University, Japan). R.J. Larson (Procter and Gamble Environmental Science Department) provided key suggestions to experimental designs and this report. This work has also benefited from SCAS unit discussions with S.K. Kaiser, M.A. Hansmann, and E.A. Bookland; and Biolog data analysis discussions with D.M. Lee; all of the Procter and Gamble Environmental Science Department.

DRESS(ROW()-1,4)),INDIRECT(ADDRESS(2, COLUMN())):INDIRECT(ADDRESS(ROW()-2, COLUMN( ),)) /2-SUMPRODUCT( $D$2: INDIRECT(ADDRESS(ROW()-2,4)),INDIRECT (ADDRESS(3,COLUMN())):INDIRECT(ADDRESS(ROW()-l,COLUMN())))/2-SUMPRODUCT($D$2:INDIRECT(ADDRESS(ROW()2,4)),INDIRECT(ADDRESS(2,COLUMN())):INDIRECT(ADDRESS(ROW( )-2,COLUMN( ))))/2

EXAMPLE: i
0

ti (time)
0

v, (value)
0

1 2 3 4

0 1 2 6

3 6 9 10

The trapezoidal

area would be:

+

C((q +
Appendix A: Excel formula for calculating trapezoidal approximation of area under curve The Excel formula below calculates a trapezoidal approximation of the area under the curve [23]. It assumes the D column is used for elapsed time and that D3 is the baseline or time of first measurement. Cell D2 will always be 0. Setting D2 to 0 assures the formulas will work correctly even if the first measurement time (D3) is not 0. The 96 columns (usually F - CW) are used for storing the observed values for each of the 96 wells at the times indicated in column D. The formula should be stored in the cell immediately below the last observation in each of the 96 columns (wells). The formula is written to be self adjusting. It automatically determines the number of measurements and automatically updates calculations when data are added. The text of the formula is the same in each of the 96 columns. Formula: =SUMPRODUCT($D$3:INDIRECT(ADDRESS (ROW()-1,4)),INDIRECT(ADDRESS(3,COLUMN ())):INDIRECT(ADDRESS(ROW()-ICOLUMN ())))/2+SUMPRODUCT($D$3:INDIRECT(ADi=l

y_,)l2)*(t;

Ll)

= 5o

The Excel formula above uses a less straightforward method that is mathematically equivalent:

References
Cl1 Bochner,
B.R. (1989) Sleuthing out bacterial identities. Nature 339. 157-158. 121Klinger, J.M., Stowe, R.P., Obenhuber, D.C., Groves, T.O., Mishra SK. and Pierson, D.L. (1992) Evaluation of the Biolog automated microbial identification system. Appl. Environ. Microbial. 58, 2089-2092. [31 Biolog. (1993) GP MicroPlate Instructions for Use. Biolog, Inc. [41 Biolog. (1993) GN MicroPlate Instructions for Use. Biolog, Inc. and PI Garland, J.L. and Mills, A.L. (1991) Classification characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl. Environ. Microbial. 57, 2351-2359. [61 Garland, J.L. (1996) Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biol. Biochem. 28, 213221.

J.B. Guckert et al. / Journal of Microbiological Methods 27 (1996) 183-197 [7] Zak, J.C., Willig, M.R., Moorhead, D.L. and Wildman, H.G. (1994) Functional diversity of microbial communities: a quantitative approach. Soil Biol. Biochem. 26, 1101-1108. [8] Haack, SK., Garchow, H., Klug, M.J. and Forney, L.J. (1995) Analysis of factors affecting the accuracy, reproducibility and interpretation of microbial community carbon source utilization patterns. Appl. Environ. Microbial. 61, 1458-1468. [9] Victoria, L., Gilbride, K.A.. Allen, D.G. and Liss, S.N. (1996) Phenotypic fingerprinting of microbial communities in wastewater treatment systems. Water Res. 30, 1077-1086. [IO] Biolog. (1993) MT MicroPlate Instructions for Use. Biolog, Inc. [ 1l] Gorden, R.W., Hazen, T.C. and Fliermans, C.B. (1993) Rapid screening for bacteria capable of degrading toxic organic compounds. .I. Microbial. Methods 18, 339-347. [12] Lee, C., Russell, N.J. and White, G.F. (1995) Rapid screening for bacterial phenotypes capable of biodegrading anionic surfactants: development and validation of a microtitre plate method. Microbiology 141, 2801-2810. [13] la Riviere, J.W.M. (1977) Microbial ecology of liquid waste treatment. Adv. Microb. Ecol. 1, 215. [14] Wagner, M., Amann, R., Lemmer. H. and Schleifer, K.-H. (1993) Probing activated sludge with oligonucleotides specific for Proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbial. 59, 1520-1525. [15] Boczar, B.A., Begley. W.M. and Larson, R.J. (1992) Charac-

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[22] Hurlbert, S.H. (1984) Pseudoreplication and the design of ecological field experiments. Ecology 54, 187-211. [23] Grossman, S.I. (1981) Calculus, Academic Press, New York. pp. 470-475.