Impacts d fl ow and multisensor-technology for microbial activity pro fi ling †

The combination of micro-segmented flow with miniaturized flow-through multisensor-technology has been utilized for metabolite profiling of soil bacteria. Series of sub-ml segments were generated containing soil sample slurry from historic copper mining sites and exposed to heavy metal salts of copper and nickel. Segments were examined for bacterial growth and spectral properties as well as for the effect of heavy metal-treatment after different incubation times. In order to evaluate microbial growth, extinction was recorded with 4 different spectral channels. Fluorescence was measured using a microflow-through fluorometer to detect both growth and production of fluorescent dyes or metabolites. The incidence of single segments with enhanced absorption in one of the spectral channels or enhanced fluorescence was scored to detect soil microorganisms with interesting properties for further screening. The study could show that the number of vegetated segments, the density of microorganisms in the segments after cultivation and the spectral response are different for separate soil samples and different metals. Thus, the highly parallelized and miniaturized segmented flow method is a promising tool for profiling of soil samples with regard to identifying micro-organisms with interesting profiles for secondary metabolite-production.


Introduction
Soils contain large numbers of microorganisms and are very diverse habitats.Most microorganisms in soils are yet unknown, with only 0.1 to 10% of soil bacteria being cultivatable 1,2 and metagenomic data from soil samples implying a huge number of undiscovered species. 3Thus, the biochemical capacity of the vast majority of soil microbes so far remains untapped, albeit an increasing necessity for new drugs has been acknowledged by, e.g.World Health Organisation.
Most soil bacteria can survive under difficult environmental conditions due to their evolutionary adaptation and their symbiotic coexistence in communities of organisms.For example, there are micro-organisms and communities that can exist in heavy metal-contaminated soils.Pollution of soils with heavy metals occurs e.g. from rapidly expanding industrial areas, abandoned mining areas, contaminated waste water or animal manures 4 and typical bacterial tolerance strategies against heavy metals include binding, complexation, accumulation, oxidation/reduction, precipitation and efflux of heavy metals. 5These bacterial tolerance mechanisms can be utilized for bioremediation of heavy-metal-contaminated soils with dead or live microbial biomass to sequester metals from contaminated areas. 6Recently, studies with such heavy metal-tolerant soil streptomycetes revealed that heavy metal-supplementation induces secondary metabolite production. 7Thus, these bio-remediating bacteria have two very advantageous properties, which make them interesting research objects.
It is well known that many soil microorganisms in particular the bacterial group of streptomycetes can produce a large variety of bioactive secondary metabolites including most antibiotics in use today and can, therefore, be considered an important source for new drugs and new biosynthetic pathways. 8Both growth and production of secondary metabolites like antibiotics, pigments, etc. are strongly dependent on cultivation conditions.Different microbial species require special media formulations for growth and induction of secondary metabolite-production. 9The screening of soil samples for unknown microorganisms and the optimization of the cultivation media for growth and induction of secondary metabolite production is a multi-dimensional problem.Therefore, traditional cultivation techniques have to be complemented by new approaches combining highly parallelized screening with minimal resource requirements, both for chemicals and space, while providing good statistics with manifold replicates.The applications of microuidic techniques with droplet-based techniques 10,11 or microuidic segments 12 are particularly well suited for such a miniaturized, multi-parameter approach.
The micro-segmented ow technique used here allows for the generation and handling of hundreds or thousands of samples in parallel, combined with an addressable order.The typical volumes are in the sub-mL and nL range.It could be shown that bacteria 13,14 and eukaryotic microorganisms 15,16 can be cultivated in these small volumes and the technique was successful for the determination of EC 50 -values of antibiotics and other chemical effectors by highly resolved dose/response functions, as well as for the determination of combinatory effects in two-and three-dimensional concentration spaces. 17eading the bacterial growth inside segments can be realized by microow-through photometry, measuring extinction which is nearly proportional to the density of bacteria.In addition, the detection of organism-derived uorescence can provide information on growth; at the same time it might indicate the occurrence of additional, uorescent metabolites produced during incubation within segments.Plant secondary metabolites have been identied already using micro-spectral uorimetric studies. 18 major advantage of the segmented ow-technique is the computer-controlled generation of high-resolution concentration gradients of chemicals in segments, or the precise dosing of different chemicals in segment sequences containing cells or micro-organisms.For application to soil, this specic segment generation might prove useful if every segment can be inoculated with soil sample slurry. 19Due to the high number of segments a very large amount of soil microorganisms can be addressed simultaneously.Random encapsulation of soilderived different microorganism species in small enclosed compartments leads to the formation of random mixed microorganism cultures with restricted cell numbers.For such mixed cultures the activation of gene promoters has been shown, which control the synthesis of secondary metabolites, which was not observed in pure cultures 20 and coculture studies with known bacterial species revealed induction or potentiation of metabolite production. 21Using segmented ow-technology different agents can be readily co-administered to stimulate growth or secondary metabolite production.3][24] For some Streptomyces producer strains (S. tendae F4, S. acidiscabies E13), the cultivation in segments and multi-heavy metal effects have been shown. 25ased on these results, we wanted to investigate different soil samples using moderate concentrations of heavy metals to stimulate secondary metabolism.It is expected that different soil samples represent different microbial communities, most likely including potential new secondary metabolite producers.A proling of soil samples by photometry and uorometry aer incubation would provide an elegant tool to select interesting microorganism-communities due to their spectral properties and would allow testing different sampling sites prior to intensive screening and isolation of pure cultures.In addition, the application of sensor sets or spectral sensing should help identifying samples with bacteria producing chromophores or uorophores under different test conditions, here provided by multi-metal stress conditions.
Here, we present a methodological approach using microsegmented ow-technology to test for growth and spectral response behavior of soil microorganisms in response to heavy metal stress.This strategy has the potential of screening signicantly larger amounts of samples, already been shown for other segmented ow-based high-throughput-applications. 26,27

Soil sample collection and preparation
Soil samples were collected from different former mining areas in Thuringia and Saxony-Anhalt, Germany.The idea behind was that multi-element contaminated soils have led to an enrichment of metal-responsive microorganisms.The contamination is due to late paleozoic Zechstein (Tartarian) and in particular copper shale mining that took place since the middle ages, with more intensive exploitation from the 15th to the 18th century. 28he suldic ores lead to acid mine drainage when weathering which accelerated metal mobility. 29All collection places, the date of collection, GPS coordinates and a short description of the sampling places are listed in Table 1.
Soil samples were taken directly from the surface of the earth using a sterile falcon tube which was immediately sealed.Then they were air-dried under sterile conditions in the laboratory.For the experiments soil samples were treated as follows: 1 g soil was mixed with 40 ml sterile Aqua dest.and vortexed.Aer centrifugation for 20 minutes at 1200 rpm the supernatant was ltered through a sterile lter paper (GE Healthcare, Germany).The ltered undiluted solution containing mainly bacterial spores and vegetative bacteria was used for the experiments aer addition of the eukaryotic translation inhibitor cycloheximide at a nal concentration of 15 mg ml À1 to prevent growth of soil-derived fungi in segments.

Experimental setup
The experimental set-up for segment generation, incubation and optical characterization of spectral properties of the segment content is shown in Fig. 1.
In detail, 500 nL segments were formed by coinjection of soil sample slurry (5 ml min À1 ), cultivation medium with heavy metal salts at a nal concentration of 0.25 mM or 0.30 mM (5 ml min À1 ) into a ow of carrier solution PP 9 (40 ml min À1 ) by using a PEEK™ 7-port manifold (YMC Europe GmbH, Dinslaken, Germany).This was done with the help of a computer-controlled syringe pump with 3 dosing-units (Cetoni GmbH, Korbußen, Germany), implemented with syringes (ILS, Stützerbach, Germany) with 500 ml (soil sample slurry and cultivation medium with heavy metals) and 5000 ml (carrier solution PP 9) volume.Connection of syringes with the manifold occurred by Teon® tubes (0.5 mm id, 1.6 mm od, Bohlender GmbH, Germany) with suitable ttings (YMC Europe GmbH).Aer formation and aer incubation segments were transported with a constant ow rate of 50 ml min À1 through an optical multisensor-detector unit for the simultaneous measurement of extinction and uorescence of the segment content.Extinction was measured with 4 LEDs with peak wavelengths of 470 nm (Nichia, Japan), 505 nm (Agilent, United States), 615 nm (Agilent) and 660 nm (Kingbright, Taiwan) each with a photodiode (Osram, Germany) for the detection of light intensity aer attenuation by microorganisms inside segments.Measurement of uorescence induction was carried out with a LED with a peak wavelength of 470 nm (ledxon, Germany) with a combination of shortpass (480 nm) and longpass lters (500 nm) (Laser Components, Germany).The emitted photons were counted using a photomultiplier module (Hamamatsu, Japan).Values for uorescence induction were normalized against the reference uorescence of the tube subtracted with 1.
Segments containing soil slurry from different collection places and different heavy metal treatments were stored in tube coils consisting of a PMMA plate with rolled transparent Tef-lon® (PTFE) tubes for 9 and 15 days in an incubator at 28 C.

Data processing-synchronization
Segment sequences were measured online at 100 Hz sampling rate shortly aer formation and aer incubation.Detection of segments occurred in a subsequent offline data analysis process using LabView™ soware and provided segment-specic data, like segment size, distance between two neighbour segments and extinction or uorescence.Segment detection was performed by the usage of three parameters: dening the upper and the lower threshold of the extinction range of the carrier liquid and dening the time interval corresponding to the minimum segment distance.Segments are recognized, when  the segment-derived extinction exceeds the thresholds of the carrier liquid.The end of the segment is detected when all extinction values in this time interval are to be found within the thresholds.Since the microorganisms grew partially inhomogeneous inside segments, growth behaviour was analysed by forming the extinction signal integral with respect to the segment size.This value is termed in the diagrams as calculated signal (calc.signal).Due to possible false detections in the individual sensor channels, time-based synchronization for unique segment mapping was conducted (Fig. S1A and B †).The rst, in all channels correctly detected segment, is used as the reference segment for time-based comparison of the following segments.False detection can occur if the extinction of the segment for the specied time interval corresponds to the extinction of the carrier liquid (increase in segment number) (Fig. S1C †) or if the distance between two segments is less than the dened time interval (decrease in segment number).
Usually, only a few false detections occur (approx.1%).These are recognized by the data synchronization and are not included in further data analysis.Thus, time-based synchronization allows unique assignment between individual segments (Fig. S1B †) and prevents analysis errors.The data obtained were then exported as tables and converted into graphics (Fig. 2).All measurement data from the individual segments were carefully compared shortly aer segment generation and aer incubation to verify that increases in the calculated signals were due to growth.Calculated signals aer incubation which were equivalent to the calculated signals of the 0 hour-measurements were designated as "non-vegetated".Calculated signals of "vegetated" segments differed considerably from the calculated signals of "non-vegetated signals".

Isolation of pigment-producing bacteria
Segmented soil sample G 17 was incubated in AM minimal medium containing 1% glucose and 0.3 mM nickel sulfate for 9 days in a Teon tube rolled on a PMAA plate at 28 C in an incubator.Subsequently, the segment sequence was measured online with the multisensor-detector unit and stored temporarily in another tube coil.Aer data analysis and time synchronization, the segment sequence was transported out of the Teon tube by constant pumping of the carrier uid.Meanwhile, segments were counted and segment no. 46 was collected in an Eppendorf tube.The segment content in the tube was mixed with 100 ml of the aforementioned medium and then transferred into 10 ml liquid medium with the same composition in an Erlenmeyer ask.This suspension was cultivated for 24 h at 28 C at 120 rpm. 10 ml of the liquid bacterial culture was plated on an AM minimal medium agar plate containing 1% glucose and 0.3 mM nickel sulfate (solid).
Investigation of heavy-metal-resistance of the bacterial isolate under segmented ow-conditions 10 ml AM minimal medium containing 1% glucose was inoculated with a colony of the pigment-producing bacterial isolate and incubated at 28 C at 120 rpm overnight.Continuous concentration gradients of nickel-and copper sulfate in segments containing the bacterial isolate G17/46 (inoculation cell number: 2500 bacteria/500 nL segment) were generated with the help of the LabView™ soware controlling the ow rate of the syringe pumps.Segments containing the bacteria, incubation medium and the heavy metals in increasing concentrations were formed by variation of the ow rates.
Increasing amounts of copper sulfate or nickel sulfate (ow rate from 0-5 ml min À1 ) were compensated by a decreasing amount of incubation medium (5-0 ml min À1 ).The nal concentration of copper sulfate and nickel sulfate in the segments was 2 mM and 5 mM, respectively.The carrier liquid PP9 was transported with a constant ow rate of 40 ml min À1 .Segments were stored for 48 h in tube coils at 28 C. Aer incubation, extinction of the segments at 505 nm was measured to determine heavy-metal tolerance of the bacterial isolate.The experiments were repeated two times.
Fractionation, ethanol treatment of soil sample and measurement of segment-derived uorescence 10 ml soil sample slurry of G 9 was inoculated into 10 ml AM minimal medium containing 1% glucose reaching a nal volume of 20 ml.This suspension was incubated for 9 days in an Erlenmeyer ask with constant slow stirring at 28 C. The rest of the soil sample slurry was cooled at 4 C in a fridge.Soil sample culture aer 9 days growth was counted to determine the cell number.10 ml of the soil sample was centrifuged for 10 min at 4500 rpm.The supernatant was transferred into a new falcon tube and the pellet was resuspended in fresh AM minimal medium 1% glucose.To obtain a dead soil sample again a 10 ml soil sample with the same cell number like before was centrifuged for 10 min at 4500 rpm and subsequently, the cell pellet was incubated for 1 h with 5 ml 70% ethanol.The cell pellet was washed 2Â with 1Â phosphate buffered saline and nally resuspended in 10 ml AM minimal medium/1% glucose.The starting soil sample, resuspended pellet (living), supernatant, resuspended pellet (dead) and AM medium/1% glucose (mixed 1 : 1 with soil sample slurry) were segmented in a ow rate ratio of 10 ml min À1 for cells or supernatant and 40 ml min À1 for the carrier liquid PP9 and the uorescence of the segments was measured aer excitation with 470 nm.Fluorescence values were normalized against the blank AM-medium/1% glucose mixed 1 : 1 with starting soil slurry solution.

FDA/PI staining
To prove whether ethanol treatment was successful in killing soil microorganisms FDA (uorescein diacetate)/PI (propidium iodide) staining was performed with living and dead resuspended soil sample-pellets like described before.Non-treated and ethanol-treated soil samples were incubated with 10 mg ml À1 PI and 50 mM FDA in phosphate buffered saline for 30 min to stain living and dead microorganisms.Images of stained soil samples were taken with a Zeiss/Axioplan using lter sets of 510/580 nm for PI-and 450/510 nm for FDA-immunouorescence.

Results and discussion
Cultivation directly from soil samples First, we wanted to nd out whether growth of microorganisms present in the soil sample can be detected by means of extinction measurements.Therefore, soil slurries were injected (without further dilution) and the segments were measured with excitation wavelengths of 470, 505, 615 and 660 nm aer 9 days growth.The presence or absence of bacterial growth could be easily detected by the intensity of the signals; extinction differed considerably from segment to segment, from sample to sample and in the presence of different heavy metals.However, all four spectral channels behaved similarly in response to growth which indicates a lack of production of chromophores.An exemplary dataset is shown in Fig. 3A where soil sample G 9 was cultivated with copper sulfate.Thus, the high correlation of the signals indicates an increase in cell number inside every segment.To verify that the measured signals aer cultivation are due to growth, calculated signals were compared for all sensor channels to the signals of the 0 hour-measurements (see description in data processing and synchronization).A comparison of sensor signals of the segment sequence from Fig. 3A at 505 nm wavelength before and aer cultivation is shown in Fig. 3B.
To further examine extinction accuracy for growth detection, correlation plots were created where the optical signals from two sensors with different excitation wavelengths (470 nm; 660 nm) were correlated with each other.Using this analysis, differential responses by different wavelength measurements can be identied which would translate into potentially interesting, well-growing isolates inside the segments.The location of the segments in the graph also reects their vegetation density.The example shown in Fig. 4A is typical for a cultivation study, where only some segments are prone to undergo growth, and the corresponding signals of the optical channels correlated with each other (see Fig. 4C).However, other soil samples showed a higher number of vegetated segments, indicating

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higher proportions of bacteria being present in this soil.There, extinction measurements correlated well with each other again (Fig. 4B and D).
The method can also be applied to investigate the growth response of microbes in a soil sample to several metals, or various soil samples to one metal.As an example, two soil samples, K 1 and K 6, were segmented and incubated either with 0.30 mM copper sulfate or 0.30 mM nickel sulfate for 9 days, and extinction signals for 470 and 660 nm were analysed by correlation plots.A similar response for different soil samples to the respective metal indicated similar toxicity and hence microbial response to the respective metal.Aer nickel sulfate-treatment both soil samples showed more vegetated segments with higher extinction-values and generally a higher scattering of extinctions compared to copper sulfate-incubated soil microorganisms (Fig. 5).However, under nickel sulfate treatment there were also found segments with lower vegetation compared to copper-treated.This might well be due to the fact that copper, as an important trace metal and component of many bacterial enzymes, slightly stimulates growth but it is also a more potent inducer of oxidative stress in microbes than nickel. 30Therefore it can be assumed that copper sulfate at this concentration stimulates but also suppresses higher growth of micro-organisms in both soil samples.In contrast, in the presence of nickel sulfate at the same concentration, segments with rising and scattering optical density indicated the presence of poor-and better growing microorganisms depending on their ability to tolerate nickel.
For a better classication and comparison of soil samples, ranking spectra were generated from the datasets.The graphs allow for a simple classication of response groups of segments in each sequence depending on the extinction signal (Fig. 6).
The ranking spectrum of soil sample K 6 reects the presence of a number of segments with low extinction (low scattering and hence low growth) and other groups of segments with increasing optical response and higher extinction indicating "good growth".In contrast, the ranking spectra of soil sample K 1 and M 7 are marked by a smooth or stepwise, respectively, increase of extinction which indicates a more uniform distribution of vegetated segments and hence can be interpreted as a population of micro-organisms with metaltolerance levels present that allow for a wider variety of metals in the droplets to still obtain growth.Segments that were inoculated with soil sample M 7 showed the highest extinction values in the presence of nickel sulfate indicating better nickel tolerance or resistance for this soil sample.Typically, all four photometric sensors showed a similar or identical type of ranking spectra, again underlining lack of chromophore production (data not shown).The number of ranks in each class of signals and their mean extinction level are specic parameters for the undiluted soil community response to heavy metal exposition.Obviously, these parameters can be used as general parameter to distinguish different soil sample-microoras under the same stress conditions.

Identication of optical indicators for secondary metabolite production
Besides a general classication of growth from soil samples, the detection of the production of secondary metabolites was of particular interest.The application of multi-sensor technology can help to identify production scenarios.Therefore, online detection with optical sensors was used.To validate our assumption, we rst searched for pigmented soil bacteria.The formation of chromophores can easily be identied by altered correlation between the photometric signals, depending on the spectral features of the coloured compound.An enhanced absorption signal of the short-wavelength sensors reects an increase of blue light absorption and hence a yellow colour of the segment content.In contrast, the enhancement of longwavelength absorption is an indicator of the formation of a brownish, red, green or even blue colour in a segment.These deviations in the correlation between different wavelengths Fig. 5 Metal tolerance of soil samples: metal tolerance of microbial communities of soil samples K 1 and K 6 was analysed by correlation plots from calculated extinction data from sensors 470 nm and 660 nm.Segment sequences were incubated for 9 days with 0.30 mM copper sulfate (A and C) and 0.30 mM nickel sulfate (B and D) in AM minimal medium/1% glucose.could be recognized from correlation diagrams.An exemplary dataset is shown in Fig. 7 where the calculated signals (extinction signal integral with respect to the segment size) of onlinemeasurements at 505 nm and 470 nm of soil sample G 17 grown under nickel sulfate-stress were correlated (Fig. 7A).Most segments did not allow for growth or were not fully grown, visible with low extinction values (e.g., see segment no. 47, Fig. 7B).Segments with strong bacterial growth were identied by high extinction values (see segment no. 57, Fig. 7C).Of particular interest was segment number 46, which plotted outside of the assumed correlation line.This was due to an enhanced absorption signal of the short-wavelength sensor at 470 nm (Fig. 7A and D) compared to the sensor with 505 nm excitation wavelength.
The subsequent inoculation of this potential chromophoreproducing microbial community into liquid solid media, followed by plating on a minimal medium agar plate revealed the growth of a strongly yellow-coloured bacterial isolate, verifying that the method can be applied for initial screening (Fig. 8A).In addition, the bacterial isolate was tested for its tolerance against the heavy metals nickel (Fig. 8B) and copper (Fig. 8C) under segmented ow-conditions.Two independent experiments revealed mean-EC 50 -values of 1.35 mM for nickel sulfate and 0.66 mM for copper sulfate-treatment showing that the tolerances of the bacterial isolate against the tested heavy metals reside in the moderate range.Thus, using multisensor-technology, a moderately heavy-metal-tolerant bacterial isolate could be gained from a larger number of soil slurry-loaded segments, which secretes a yellow pigment as secondary metabolite, most likely, to defend itself against nickel-associated oxidative stress.

Monitoring uorescence
The presence of uorophores may even better indicate secondary metabolite production.While some antibiotics are known to be coloured (e.g., actinorhodin), 31 many drugs (including antibiotics) consist of uorescent molecular structures. 32,33Since primary metabolites are known to include UV-  uorescent metabolites like NAD(P)H, avins and amino acids tryptophan and tyrosine, the uorescence measurements will also allow for well detectable signals indicating growth at low cell densities by autouorescence. 34hus, we measured uorescence signals (excitation wavelength: 470 nm) of the segments containing different soil inocula (excitation wavelength: 470 nm).Fluorescence diagrams as well as correlation plots were created.Soil samples treated with different heavy metals showed varying degrees of uorescence (Fig. 9A and B).
Comparison of uorescence diagrams for soil sample G 9 treated with copper-or nickel sulfate at 0.3 mM concentrations revealed that copper induces a higher degree of uorescence in segments than nickel correlated with the higher toxicity observed earlier.Theoretically, this could be attributed either to more uorescent cells or, more likely, to a stronger induction of (secondary) metabolites.To nd out where the measured uorescence originates, experiments with soil sample G9 in conventional ask-culture were performed.Separation of soil sample culture into particulate and supernatant fraction aer 9 days incubation revealed that almost all the uorescence is derived from the supernatant (Fig. 9B).This nicely shows that secreted metabolites of soil microorganisms must be uorescent in some way.Pelleted microorganisms from the soil sample only showed a little uorescence which can be referred here as autouorescence.To verify whether dead cells are uorescent, a "living" soil pellet was treated with 70% ethanol to produce cell death.This pellet of "dead" soil microorganisms showed the lowest uorescence.It can therefore be assumed that the measured uorescence in segments is derived mostly from secreted metabolites, but a smaller proportion also stems from living and dead microorganisms.Live/dead staining was performed to see the amount of live and dead microorganisms in the differently treated soil pellets.In the "dead" soil sample (aer ethanol treatment) FDA-staining was completely absent and only propidium iodide-staining for the detection of released DNA from dead cells could be detected.In the nontreated soil sample living as well as dead cells could be found with live/dead staining (Fig. 9C).
The potential of multi-sensor-technology for classication of soil communities in response to stress under given conditions is well reected by correlation plots for soil sample M 7 under nickel exposition (Fig. 10).The photometric signals at 660 nm and 470 nm are well correlated (Fig. 10A).However, high scattering was observed at higher extinction values.If uorescence levels are considered, segments can be subdivided into three groups representing high, moderate and low uorescence at low overall extinction levels (Fig. 10B).Thus, four types of response groups could be identied.

Conclusion
In the current study, an attempt was made to characterize soil samples from different metal-contaminated sampling sites with respect to spectral properties by means of multi-sensor technology.For this purpose, a high number of segments containing soil sample slurry incubated at known heavy metal concentrations were analysed.Growth of microbes in segments could easily be detected by measuring extinction with good correspondence between all four wavelengths.Correlation plots could show that microbial communities of different origin show similar behaviour with respect to metal toxicity.Additional information could be obtained when analysing over-representation of extinction at one wavelength.Moreover, the potential for the discovery of secondary metabolite-producing microorganisms in segments was demonstrated by the detection and isolation of a yellow-pigmented bacterial isolate which showed tolerances against nickel (EC 50 : 1.35 mM) and copper (EC 50 : 0.66 mM).Thus, it could be shown that the segmented ow technique and multisensor-technology are suited for the evaluation of physiological activity of microbial communities in the presence of heavy metals and therefore of their bioremediation potential.Monitoring of uorescence induction in the soil  sample may be used as an additional approach to detect secondary metabolites.Analysis of a long term-cultivated soil sample revealed the strongest uorescence for the metabolitecontaining culture medium.Further studies are necessary to verify that the observed uorescence is associated with an induction of secondary metabolite-production, in our case by exerting stress through addition of heavy metals.In the future our miniaturized strategy will be used for the characterization of a larger number (100-1000) of soil samples from historical mining areas with different environmental conditions.

Fig. 2
Fig. 2 Data processing: online-measurement of segment sequences and offline-data analysis with LabView™ software.

Fig. 3
Fig. 3 Multisensor-measurement: (A) synchronized data from an online-measurement of a segment sequence of 71 segments, in which soil sample G 9 was incubated with 0.25 mM copper sulfate for 9 days in AM minimal medium/1% glucose.A multi-channel photometer set with excitation wavelengths of 470, 505, 615 and 660 nm was used.Every dot in the graph represents a segment.(B) Sensor signals (excitation 505 nm) of the same segment sequence measured at 0 hours and after 9 days were compared.

Fig. 4
Fig.4Multisensor-measurement and correlation: synchronized data of online-multisensor-measurements of segment sequences of (A) soil sample G 7 treated with 0.25 mM copper sulfate and of (B) soil sample G 17 treated with 0.25 mM nickel sulfate for 15 days in AM minimal medium/1% glucose and the corresponding correlation plots for sensor 470 nm and 660 nm (C and D).Every dot in the graph represents a segment.

Fig. 6
Fig.6Analysis of soil sample-derived growth of microorganisms by extinction ranking spectra: extinction ranking spectra prepared from data of online-multisensor-measurement (470 nm) of soil samples K 1, K 6 and M 7 treated with 0.3 mM nickel sulfate for 9 days in AMminimal medium/1% glucose.

Fig. 7
Fig. 7 Optical indicator of pigment production: (A) correlation plot indicates growth response of soil sample G 17 incubated with 0.30 mM nickel sulfate for 9 days in AM minimal medium/1% glucose and the formation of a dyed product in a single segment (no.46); (B-D) extinction diagrams for single segment-photometric measurements: (B) non-vegetated segment no. 47, (C) vegetated segment no. 57 without optical conspicuousness, (D) vegetated and pigment-bearing segment no. 46.

Fig. 8
Fig.8Isolation of pigment-producing and heavy metal-tolerant bacteria: (A) after data analysis segment 46 was isolated by counting of the segment sequence of soil sample G 17 after incubation with 0.3 mM nickel sulfate for 9 days.The segment content was transferred into liquid AM minimal medium followed by plating on an AM minimal medium agar plate (solid) containing 0.3 mM nickel sulfate.Microscopic analysis showed rod-shaped bacteria.(B) and (C) Metal tolerance of isolated bacterial microorganisms.

Fig. 9
Fig. 9 Detection of soil sample-derived fluorescence in segments by micro-flow-through fluorometry at 470 nm: (A) segment sequence inoculated with soil sample G 9 was incubated with 0.25 mM copper sulfate and with 0.25 mM nickel sulfate and segment-derived fluorescence was measured.(B) Fluorescence of segments filled with soil sample completely, living soil cells (pellet), incubation medium (supernatant) or dead soil cells (70% ethanol) is shown.(C) Fluorescein diacetate/propidium iodide immunofluorescence staining of the nontreated soil cell pellet and 70% ethanol-treated soil sample pellet.

Fig. 10
Fig. 10 Correlation of sensor signals (A) correlation plot of calculated extinction signals at 470 and 660 nm shows a uniform distribution and growth of microbial communities in segments of soil sample M 7 incubated with 0.30 mM nickel sulfate for 9 days, (B) controversially, correlation of fluorescence signals with extinction signals indicates non-uniformity.

Table 1
Soil sample description