Dynamics of biofilm formation during anaerobic digestion of organic waste

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    Article history:Received 15 May 2013Received in revised form7 November 2013Accepted 27 November 2013

    Bacteria and archaea involved in the methane production duringanaerobic digestion could attach to biolm carriers and form

    isms, attached to ac substances (EPS)um [2]. The struc-layers of scatteredscopic dimensions

    three stages: thetion of the biolm

    planktonic cells. The biolm mode of life is a feature common tomost microorganisms in natural habitats [2]. Biolms are ubiqui-tous in almost every aqueous interface, such as solideliquid or aireliquid interfaces [4]. In most instances where biolms are anuisance, the term microbial fouling or biofouling is widely used[5]. For example, biofouling can be a problem in the food industry, itcontributes to human infections [6] and it can lead to biocorrosion[7]. However, biolms do not only reveal negative effects. The

    * Corresponding author. Ulm University, Institute of Microbiology and Biotech-nology, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Tel.: 49 731 5022713.

    E-mail addresses: susanne.langer@uni-ulm.de (S. Langer), daniel.schropp@uni-ulm.de (D. Schropp), frank.bengelsdorf@uni-ulm.de (F.R. Bengelsdorf), maazuza.

    Contents lists availab

    Anaer

    journal homepage: www.else

    Anaerobe xxx (2013) 1e8othman@rmit.edu.au (M. Othman), marian.kazda@uni-ulm.de (M. Kazda).tion of biolm carriers (e.g. plant material) to the biogas reactors. to complex microcolonies and the cell dispersal of highly motile1. Introduction

    The production of biogas provides a versatile carrier of renew-able energy, as methane can replace fossil fuels partly in both heatand power generation and as vehicle fuel [1]. Besides technicalimprovements of biogas plants the efciency of the biogas processcan be further improved by engineering the microbial community.

    A possible approach to improve the biogas process is the addi-

    biolms. Biolms are assemblages of microorgansurface and encased in an extracellular polymerimatrix, that functions as a cooperative consortiture of microbial communities ranges frommonosingle cells to thick, mucous structures of macro[3].

    The biolm life cycle can be divided intoattachment of single cells to a surface, the matura 2013 Elsevier Ltd. All rights reserved.Available online xxx

    Keywords:Anaerobic biolmBiolm formationAnaerobic digestionBiogas1075-9964/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.anaerobe.2013.11.013

    Please cite this article in press as: Langer S, ehttp://dx.doi.org/10.1016/j.anaerobe.2013.11a b s t r a c t

    Biolm-based reactors are effectively used for wastewater treatment but are not common in biogasproduction. This study investigated biolm dynamics on biolm carriers incubated in batch biogas re-actors at high and low organic loading rates for sludge from meat industry dissolved air otation units.Biolm formation and dynamics were studied using various microscopic techniques. Resulting micro-graphs were analysed for total cell numbers, thickness of biolms, biolm-covered surface area, and thearea covered by extracellular polymeric substances (EPS).Cell numbers within biolms (1011 cells ml1) were up to one order of magnitude higher compared to

    the numbers of cells in the uid reactor content. Further, biolm formation and structure mainlycorrelated with the numbers of microorganisms present in the uid reactor content and the organicloading. At high organic loading (45 kg VS m3), the thickness of the continuous biolm layer rangedfrom 5 to 160 mmwith an average of 51 mm and a median of 26 mm. Conversely, at lower organic loading(15 kg VS m3), only microcolonies were detectable. Those microcolonies increased in their frequency ofoccurrence during ongoing fermentation. Independently from the organic loading rate, biolms wereembedded completely in EPS within seven days. The maturation and maintenance of biolms changedduring the batch fermentation due to decreasing substrate availability. Concomitant, detachment ofmicroorganisms within biolms was observed simultaneously with the decrease of biogas formation.This study demonstrates that biolms of high cell densities can enhance digestion of organic waste and

    have positive effects on biogas production.RMIT University, Institute of Civil, EnvironmenbUlm University, Institute of Microbiology and Biotechnology, Albert-Einstein-Allee 11, 89081 Ulm, Germanyc tal and Chemical Engineering, Melbourne, Vic 3001, AustraliaMolecular biology, genetics and biotechnology

    Dynamics of biolm formation during aof organic waste

    Susanne Langer a,b,*, Daniel Schropp a, Frank R. BenMarian Kazda a

    aUlm University, Institute of Systematic Botany and Ecology, Albert-Einstein-Allee 11, 8All rights reserved.

    t al., Dynamics of biolm form.013erobic digestion

    lsdorf b, Maazuza Othman c,

    Ulm, Germany

    le at ScienceDirect

    obe

    vier .com/locate/anaerobeation during anaerobic digestion of organic waste, Anaerobe (2013),

  • aeroapplication of biolms can be found in many anaerobic systems,especially in the disposal of organic material (e.g. in sewage treat-ment or biogas production). In wastewater treatment, biolms playan important role as they create the basis of diverse aerobic andanaerobic reactors [8]. Biolms contribute to a more efcientdegradation of organic substrates and to a higher biogas ormethane yield. Moreover, biolm formation can result in a morestable degradation process. There are several explanations for thesepositive effects of biolms on anaerobic digestion. Microorganismsattach to surfaces and build up complex aggregates. Thereby, thebiomass increases, due to higher cell densities within the biolms.Thus, more efcient degradation of organic substrates is shown[9,10]. For instance, Zak [9] demonstrated that the addition of aplant-based biolm carrier improves biogas formation. The specicmethane yield and the organic drymatter degradation increased byup to 7% and 10%, respectively, due to the microbial biomass on thebiolm carriers.

    The biolm mode of life offers advantages like syntrophic in-teractions due to the physical vicinity of microorganisms withinbiolms. Syntrophism is a special case of cooperation between twometabolically different types of microorganisms, which depend oneach other for degradation of a certain substrate, typically throughtransferral of one or more metabolic intermediate(s) between thepartners [11]. Due to syntrophic interactions, the pool size of theshuttling intermediate can be kept low, resulting in an efcientcooperation [12]. Further, microorganisms attached to a biolmcarrier form an EPS matrix that offers protection. This EPS matrixprovides mechanical stability and serves as a diffusion barrier [13].The matrix entraps extracellular enzymes, and prevents the washoff of these enzymes improving the efciency of substrate degra-dation [14]. The diffusion barrier also prevents the entry of harmingsubstances into the biolm. Thus, cells within biolms are lessstrongly affected than suspended cultures from changes in envi-ronmental conditions such as temperature, pH, nutrient concen-trations, metabolic products and toxic substances [15,16]. Thosesubstances can be introduced by substrate addition or producedduring anaerobic digestion [17].

    The aim of this study was to investigate the dynamics of biolmformation in respect to different organic loading rates. Therefore,biogas reactors with low and high organic loading rates were setup. Biolm carriers were incubated in these biogas reactors andremoved after certain periods to investigate the biolms. Moreover,cell numbers within the formed biolms and uid reactor contentsof the biogas reactors were quantied. The formation of biolms isinuenced by different factors like genotypic and physico-chemicalfactors [3]. Consequently, substrate composition greatly inuencesthe biodiversity, physiology and structure of biolms [18]. Thus, thebiolm structures were also investigated in respect to differentorganic loading rates. Moreover, biogas production of biogas re-actors were measured and compared with biolm formation anddevelopment of cell numbers within biolms and uid reactorcontents.

    2. Materials and methods

    2.1. Experimental set up

    Two lab-scale biogas reactors with different organic loadingswere set up. The lab-scale biogas reactor with a high organicloading (H-OL, 12 L) was set up at Ulm University (Germany) andcontained 8 L inoculum from a full-scale biogas reactor suppliedwith swine manure, food leftovers, stale bread, corn silage andpotato peelings [19]. H-OL was fed with 2 L dissolved air otation(DAF) sludge collected from slaughterhouse wastewater (Ulmer

    S. Langer et al. / An2Fleisch GmbH, Ulm, Germany). The organic loading amounted to

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.01345 kg VS m3 (VS, volatile solids). The reactor was incubated in awater bath at 38 C and mixed every 15 min for 3 min at 60 rpm byan agitator. During fermentation, biogas production and methaneformation were measured by a Milligascounter (Dr. Ing. RitterApparatebau GmbH & Co. KG, Germany) and a methane sensor(BlueSens gas sensor GmbH, Germany) as described by Schropp[20].

    The lab-scale biogas reactor with a low organic loading (L-OL,0.5 L) was set up at RMIT University (Melbourne, Australia) with0.28 L anaerobic digested sludge from a municipal wastewatertreatment plant (Melbourne, Australia). L-OL was fed with 0.12 L ofDAF sludge. The organic loading amounted to 15 kg VS m3. Thereactor was operated at 35 C and not mixed. Biogas production ofreactor L-OL in batch experiments was measured volumetricallywith a gas burette as described by Prochzka et al. [21].

    Special biolm carriers made from polypropylene (PP) foil wereused for microscopical analysis of biolm characteristics. PP-discs( 9 mm) were punched out of a polypropylene foil ( 0.5 mm)and a hole was made in the middle of each PP-disc to slide severalPP-discs on a stainless steel wire ( 1 mm) with a length of 20 cm.One end of the wirewas formed to a loop in order to x a nylon lineto hang the biolm carrier in the reactor and to enable an easyremoval. The PP-discs were rinsed with double distilled water andethanol 70% to remove particles and were autoclaved for 20 min at120 C prior addition to reactors. These biolm carriers wereincubated in the biogas reactors for certain periods (H-OL: 1e7days; L-OL: 1e28 days).

    2.2. Fixation of samples

    In order to determine total cell numbers, samples from uidreactor contents (frc) were xed according to the protocol of Daimset al. [22]. Therefore, 0.5 ml of frc was mixed with 1.5 ml para-formaldehyde solution (4%) [23]. After 4 h of xation samples werecentrifuged at 5000 g for 3 min, the supernatant was removedand the cell pellet washed using 2 ml of phosphate buffered saline(PBS) [23] to remove the toxic paraformaldehyde and substrateresidues. This step was repeated three times. Finally, cell pelletsweremixed with 0.5 ml PBS solution and 0.5 ml ethanol (100%) andstored at 20 C.

    Biolms attached to PP-discs were removed from the reactorsand xed in FPA solution (100 ml formalin, 100 ml propionic acid,1800 ml ethanol (70%)) for one day to ensure stable xation.Further processings of those samples was dependent on the sub-sequent microscopic techniques.

    2.3. Sample preparation and microscopy

    2.3.1. Epiuorescence microscopyTotal cell numbers of microorganisms in frc and in biolms

    attached to PP-discs were analysed by epiuorescence microscopy.Therefore, xed cells were scratched of the PP-discs. Samples werediluted in PBS. Due to the aggregation of microorganisms thesamples were homogenised by either using a RiboLyser (HybaidLtd., Middlesex, UK) or a grinder and sterile glass beads ( 0.1 mm).20 ml of the homogeneous cell suspension was dropped onto eachwell of a Teon-coated slide (8 wells, 6 mm; Menzel GmbH & Co.KG, Germany) and dried for 15 min at 60 C. In order to x cells, theslidewas pulled through the ame of a Bunsen burner for 1e2 s andfurther dehydrated in 50, 80 and 100% ethanol for 3 min each time.Cells were stained with 20 ml of 1 SYBR Gold Nucleic Acid GelStain (Invitrogen GmbH, USA) per well for 10 min in the dark atroom temperature, ushed with cold double-distilled H2O, andimmediately dried with compressed air. Before microscopy, two

    be xxx (2013) 1e8drops of Citiuor AF1 (Citiuore Ltd., UK) were applied to the

    ation during anaerobic digestion of organic waste, Anaerobe (2013),

  • Light microscopic images of semi cross sections of biolms wereanalysed by ImageJ. The thickness of biolms in light microscopicimages was measured in regular intervals of 100 mm. The per-centage of surface area covered by EPS and surface area covered bybiolm in CSEM and ESEM images of biolms attached to PP-discswere also estimated by ImageJ.

    3. Results

    3.1. Total cell numbers: uid reactor contents vs. biolms

    Total cell numbers in the uid reactor contents of lab-scale

    aerobe xxx (2013) 1e8 3slide, and a cover slip was positioned to cover all wells. The uo-rescence signals of samples from reactor H-OLwere detected by theLeitz DMRBE epiuorescence microscope (Leica MicrosystemsGmbH, Germany) and uorescence signals of stained samples fromreactor L-OL was detected by the Leica DM 2500. Filter I3 (excita-tion lter: 450e490 nm, dichromatic mirror: 510 nm, suppressionlter: 515 nm) was used for both microscopes to detect SYBR Goldstained microorganisms. Epiuorescence images of samples fromthe reactor H-OLwere taken by digital camera Type DFC420C (LeicaMicrosystems GmbH, Germany) at an exposure time of 150 ms and300 ms. Epiuorescence images of samples from reactor L-OL weremade by a Nikon Digital Sight DS-SMc (Nikon Instruments Inc.,Japan) at an exposure time of 1 s.

    2.3.2. Light microscopyTo analyse the thickness of biolms, PP-discs with attached

    biomass were xed and crosscut. After xation, PP-discs withattached biolms were washed three times for 5e10 min in PBS. Asecond xationwas made in an aqueous osmium tetroxide solution2% for 1e2 h. Samples were dehydrated in 30, 50, 70 and 90%ethanol for 2e3 min. Thereafter, samples were embedded in epoxyresin at 60 C for 48 h. After polymerisation of the epoxy resin andtoluidine blue staining, semi cross sections were sliced by using amicrotome. The cross sections were xed on an object slide and acover glass was placed on the top. The cross sections were observedwith the microscope Leitz DMRBE (Leica Microsystems GmbH,Germany). Light microscopic images of the cross sections to mea-sure biolm thickness were made with a digital camera TypeDFC420C (Leica Microsystems GmbH, Germany).

    2.3.3. Conventional scanning electron microscopyThe conventional scanning electron microscope (CSEM) DSM

    942 (Carl Zeiss AG, Germany) was used in high vacuum mode forhigh resolution visualisation of biolms. After xation of biolmsattached to PP-discs samples were dehydrated for one day in 80%,90% ethanol and 100% isopropyl alcohol, respectively. Samples werefurther dehydrated by critical point drying (Polaron E 3000, PolaronEquipment Limited, England) and gold coated, subsequently. Bio-lms attached to PP-discs were visualised by CSEM DSM 942 inhigh vacuum mode. The resolution under this mode reaches up to4 nm at 30 kV. Signalling electrons were detected by a SecondaryElectron Detector (SED) and visualised on monitor. Optimal qualityof micrograph images were reached at a high voltage of 5e10 kV, apressure of 2107 hPa, aworking distance of 7e12mm and a spotsize of 9.

    2.3.4. Environmental scanning electron microscopyThe environmental scanning electron microscope (ESEM) FEI

    Quanta 200 (FEI Company, USA) was used for high-resolution vis-ualisation of biolms sampled from reactor L-OL. For visualisationof biolms no preparation was needed. ESEM was operated in wetmode (extended vacuum mode) that complied a range of pressurein the chamber from 0.1 to 26 hPa. The resolution under this modereaches up to 3 nm at 30 kV. Signalling electrons were detected by aGaseous Secondary Electron Detector (GSED) and visualised on amonitor. Optimal quality of ESEM images was reached at a highvoltage of 15 kV, a pressure of 4.5 hPa, a working distance of 5e7 mm and a spot size of 4.

    2.4. Image analysis

    Stained microorganisms in epiuorescence images were auto-matically enumerated by the software LAS Image Analysis a part ofthe Leica Application Suite V3 (Leica Microsystems GmbH, Ger-

    S. Langer et al. / Anmany) and Mac Biophotonics ImageJ (Wayne Rasband, Freeware).

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013biogas reactors with a high (H-OL, 45 kg VS m3) and a low (L-OL, 15 kg VS m3) organic loading were estimated during anaerobicdigestion over a period of 28 days (Table 1).

    In the uid reactor content of the biogas reactor with a highorganic loading 3 1010 cells ml1 were found in average. Cellnumbers in the uid reactor content of reactor H-OL were stableduring the whole fermentation process. In comparison, the reactorL-OL with a lower organic loading showed an average cell numberof 0.97 1010 cells ml1 uid reactor content, two-thirds less thanthe cells in reactor H-OL. Moreover, cell numbers in the uid reactorcontent of reactor L-OL were not stable during the whole fermen-tation process but decreased slightly after 21 days of anaerobicdigestion.

    Total cell numbers of biolms formed during the incubation ofbiolm carriers in reactor H-OL or L-OL were estimated after sevendays of anaerobic digestion. At the same time point, samples fromthe uid reactor contents were collected and the cell numbers wereestimated by epiuorescence microscopy. The cell numbers withinthe biolms and the uid reactor contents were compared to eachother (Table 1). Total cell numbers within the biolms(1011 cells ml1) were one order of magnitude higher compared tothe numbers of cells in the uid reactor content (1010 cells ml1).Additionally, cell numbers within the biolms of reactor H-OL wereslightly higher compared to cell numbers within biolms of reactorL-OL.

    3.2. Biolm surface structure

    The structure of biolms can be inuenced bymany factors. Oneof these factors can be the amount of the available substrate. Highorganic loading rates contributed to a continuous biolm layer. Athin layer of reactor content immediately covered biolm carriersincubated in H-OL. Conversely, at lower organic loading ratesmicrocolonies were detectable. They showed a unique architectureand structure (Fig 1). Structures ranged from single scattered mi-croorganisms and thin uncontinuous layers up to complex formswith different shapes embedded in an EPS matrix. Most of themicrocolonies were semi-spherical. Some of them were column-shaped or showed a tulip-like architecture others showed dense

    Table 1Total cell numbers per ml uid reactor content from lab-scale biogas reactors set upwith high (H-OL, 45 kg VS m3) and low organic loadings (L-OL, 15 kg VS m3).

    Time [d] H-OL L-OL

    Cells ml1 uid reactor content0 (4.42 1.93) 1010 (na 30) (1.20 0.32) 1010 (n 10)7 (2.64 0.51) 1010 (n 30) (1.07 0.63) 1010 (n 10)14 (3.38 1.27) 1010 (n 30) (1.01 0.27) 1010 (n 10)21 (2.09 0.54) 1010 (n 30) (0.76 0.23) 1010 (n 10)28 (2.43 0.72) 1010 (n 30) (0.61 0.20) 1010 (n 10)Cells ml1 biolm7 (2.96 1.05) 1011 (n 20) (2.09 0.99) 1011 (n 10)

    a n number of analysed epiuorescence images.

    ation during anaerobic digestion of organic waste, Anaerobe (2013),

  • Fig. 1. ESEM images; microcolonies attached to biolm carriers incubated for 21 days in reactor at low organic loadings; scale bar 100 mm.

    S. Langer et al. / Anaerobe xxx (2013) 1e84knobbly structures. In particular, semi-spherical microcoloniesappeared to be uffy on the surface.

    3.3. Biolm formation and biogas production

    The formation of biolms is a dynamic process that depends onmany different factors. The thickness of biolms was measuredonly at high organic loadings, mainly because the uncontinuousmicrocolony formation at low organic loadings did not allowmeasurement by the available method. As described before, at highorganic loadings biolms appeared in the form of a continuouslayer. During microscopy it became obvious that plant and otherunidentied particles within the uid reactor content of the biogasreactor mainly contributed to the thickness of the biolm layer (Fig2). Although, the biolm layer was continuous it showed greatsurface irregularities. Consequently, there was no remarkable in-crease of the biolm thickness noticed within the observation timeof seven days. Moreover, the measured values of the biolmthickness showed great variations, also within samples collected atthe same time point (Fig 3). The thickness of the biolms sampledfrom reactor H-OL ranged from 5 to 160 mm. Thus, the average of allmeasurements was 51 mm, whereas the medianwas only 26 mm. Onaverage there was no signicant increase in biolm thicknessmeasurable during the time of observation.

    At low organic loadings microcolonies were formed instead of acontinuous biolm layer. These microcolonies were not detectableafter one day (Fig 4.1), but were rst recognised after three days ofincubation. The number and size of those colonies increased withinthe 21 days, and occurred most frequently during day 14e21 (Fig4.2). Their average diameter was 52 mm after 14 days and 93 mmafter 21 days. Thereafter, a dispersal of cells and a detachment ofmicrocolonies were observed during analysis (Fig 4.3).

    At a low organic loading the biolm-covered surface area andtotal cell numbers per cm2 biolm (Fig 5.1) were investigated for aFig. 2. Light microscopic images; semi cross section

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013period of 28 days. Since at low organic loadings, the biolm carrierwas not completely covered by a biolm layer, changes in thebiolm growth were measurable by the biolm-covered surfacearea. After one day of incubation, only 14% of the biolm carrier wascovered by biolm. The biolm covered surface area increased up to51% after 21 days of incubation and dropped to 13% after 28 days.

    Total cell numbers per cm2 biolm carrier showed a similardevelopment. After one day of incubation at low organic loadings,1.74 107 cells cm2 were detectable. In the rst seven days ofanaerobic digestion cell numbers per cm2 biolm doubled to3.55 107 cells cm2 and stayed stable until day 21 of anaerobicdigestion. After 21 days, cell numbers dropped to1.17 107 cells cm2. In the period of 21 days, when biolm growthwere detectable in respect to microcolony formation, biolmcovered surface area and cell numbers per cm2 biolm carrier, anincrease in biogas production was measured. Interestingly, at thesame time, biolm formation decreased and cell dispersal becamevisible in the form of hollow microcolonies the biogas productiondecreased as well. Statistical analysis showed a strong correlationbetween biolm development and biogas production at loworganic loading rates (Spearman R 0.8, N 5, r < 0.01).

    At a high organic loading the biolm covered surface area andtotal cell numbers per cm2 biolm (Fig 5.2) were investigated for atime period of seven days. The biolm carrier was coveredcompletely by the reactor content, due to its high viscosity at highorganic loading rates. Consequently, changes in biolm growthwere not measurable by the biolm covered surface area. Thebiolm covered surface area was 100% during the whole observa-tion period of seven days. Total cell numbers per cm2 biolmcarrier increased during anaerobic digestion. After one day of in-cubation, 0.76 108 cells cm2 biolmwere detectable. After sevendays of anaerobic digestion, the cell numbers doubled to1.5 109 cells cm2 biolm similar to cell numbers at low organicloading rates. Since the biolm formation was investigated onlys of a biolm with incorporated plant particles.

    ation during anaerobic digestion of organic waste, Anaerobe (2013),

  • incubation (Fig 6.1) and increased to an almost continuous layer

    aerobe xxx (2013) 1e8 5during further anaerobic digestion independently from the organicloading (Fig 6.2).

    4. Discussion

    The dynamics of biolm formation were investigated duringanaerobic digestion. Total cell numbers within biolms and uidreactor contents of the biogas reactors with different loading rateswere determined. In general, total cell numbers in the uid reactorcontents of the experimental reactors fed with DAF sludge were inthe range of 1010 cells ml1. Similar results were previously re-over a period of 7 days no connection of biolm formation to thebiogas production could be made (Fig 5.4).

    Microorganisms within biolms were embedded in an EPSmatrix that consisted mainly of polysaccharides. In ESEM images ofbiolms, the EPS matrix was fully hydrated and covered microor-ganisms completely. In contrast, in CSEM images, the dehydratedEPS matrix occurred as a netlike structure due to sample prepara-tion. These EPS structures appeared immediately after one day of

    Fig. 3. Thickness of biolms [mm] at a high organic loading rate over a period of 7 days.

    S. Langer et al. / Anported by Bengelsdorf et al. [24] and Krakat et al. [25]. In contrast,cell numbers within biolms were one order of magnitude higherin the range of 1011 cells ml1. Most likely the mentioned positiveeffects of biolms on the biogas process resulted from the high celldensity and physical vicinity within biolms, which allowed forsyntrophic interactions [12].

    Cell numbers at high organic loading rates were three timeshigher compared to cell numbers at low organic loading rates(Table 1), but were still in the same order of magnitude. The highersubstrate availability and a greater amount of surface area due tosolids, which allow biolm formation, might explain this slightdifference.

    Besides cell numbers within the uid reactor contents, biolmstructure was also correlated to the organic loading (Fig 1). Further,the structure of biolms is correlated to the attachment phase [26].The initial attachment of cells to a biolm carrier is entirely randomand depends on what lands where and when [3]. Moreover,different structures of biolms are mainly inuenced by substrateconcentration [3]. In this study, the structure of biolmswas widelydependent on the starting conditions of reactors such as organicloading and total numbers of microorganisms in the uid reactorcontents. High organic loadings and the associated high cellnumbers in the uid reactor content led to the formation of a

    Fig. 4. ESEM images; biolm formation on biolm carriers at low organic loadingsafter 1 day (1), 14 days (2) and 21 days (3). Biolm formation after one day of incu-bation showed no microcolonies (1). After 7e14 days microcolonies attached to biolmcarriers were detectable. Biolm carriers incubated for more than 14 days (3) showedsallied and hollow microcolonies as indicated by the arrow. Scale bar 100 mm.

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formation during anaerobic digestion of organic waste, Anaerobe (2013),http://dx.doi.org/10.1016/j.anaerobe.2013.11.013

  • loads an[cel

    aerocontinuous biolm layer. In contrast, lower organic loadingsresulted in microcolony formation. This type of biolm structure isnamed the heterogeneous mosaic model [27]. These micro-colonies showed complex architectures and surface structures.These unique structures of the microcolonies indicate that differenttypes of microorganisms were involved in the biolm formation,since the microorganisms have a marked effect on the structure[26].

    Although a continuous biolm layer was formed at high organicloadings, biolms showed irregularities. As a consequence, thethickness of the biolms showed great variations and ranged from5 to 160 mm at high organic loadings. On the one hand, plant par-ticles contributed to the irregularities; on the other hand manyother authors [3,24,26] pointed out that biolms are highly het-erogeneous structures. These irregularities in the structure of bio-lms are not only characteristic for multispecies biolms, but alsofor pure culture biolms [26]. Further Walker et al. [27] described a

    Fig. 5. Biolm formation during anaerobic digestion at low (1, 3) and high (2, 4) organicproduction of the biogas reactors. The results were displayed over a period of 28 day[cells cm2], ( ) cells per ml biolm [cells ml1], ( ) cells per ml uid reactor content

    S. Langer et al. / An6biolm as a non-uniform structure with a variable thickness thatcan change signicantly over distances of 10 mm or less [28].Therefore, the thickness of biolms was not a suitable parameter toanalyse biolm formation during anaerobic digestion. One of themodel organisms for biolm development is Pseudomonas aerugi-nosa. In comparison to the results of this study, other researcherscharacterised the variability in thickness of P. aeruginosa biolms.For example, the mean of the thickness of a biolm formed byP. aeruginosa was 33 mm, with a range of 13.3e60 mm [29].

    The dynamics of biolms were analysed in terms microcolonyformation, biolm covered surface area and total cell numbers inrespect to different organic loading rates. The biolm formation ofthe biogas reactor with a low organic loading was observed over aperiod of 28 days. In summary, the size and frequency of micro-colonies, the biolm covered surface area and cell numbers per cm2

    biolm carrier area increased markedly during the rst 7 days ofanaerobic digestion. Thus, results indicate the attachment andconsolidation phase of the biolm life cycle. Cell numbers per cm2

    were in the range of 107e108 cells cm2.Within the following 14 days of fermentation cell numbers per

    cm2 biolm carrier and the biolm carrier surface area increasedslightly or were stable, whereas microcolonies appeared moreoften and different structures of those became visible. These fea-tures are characteristic of the maturation phase of biolms indi-cated by reduced growth rates and development of unique

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013structures [26]. The biolm mode of life is restricted for bacterialgrowth and the transcriptome of mature biolm, on average, re-sembles that of stationary-phase cells [30]. The last stage in thedevelopment of biolms is the detachment and dispersal of mi-croorganisms. Microorganisms break the biolm bond by differenteffectors such as enzymes or bacteriophages [2]. After 21 days ofanaerobic digestion dispersal of cells became visible in the form ofhollow microcolonies. It is known that the formation of hollowmicrocolonies occur when microorganisms evacuate the micro-colonies to disperse for a new habitat [2]. This is a kind of an activeand dramatic form of dispersal, sometimes referred to as seedingdispersal [31]. In this process, the surface-attached microcoloniesof ageing biolms undergo internal disintegration, leaving behindhollow shell-like structures [31]. Furthermore, cell number percm2 and the biolm covered area decreased. The dispersal of mi-croorganisms from biolms can be inuenced by many factors,especially alterations in the habitat and environmental quality,

    ing rates compared to total cell numbers within the uid reactor content and the biogasd 7 days, respectively. ( ) Biolm covered surface area [%], ( ) cells per cm2 biolmls ml1], ( ) biogas production rate [Nl kg1 VS1 d1].

    be xxx (2013) 1e8such as temperature, pH or nutrient availability [2]. In this exper-iment, the most probable reason for active detachment was alimitation of substrate supply.

    In addition to the biolm formation the biogas production ofbiogas reactors was analysed during anaerobic digestion and linkedto the biolm development. At low organic loadings biolmdevelopment was correlated to biogas production. Biogas accu-mulated within the rst 21 days of anaerobic digestion anddecreased afterwards. When dispersal of cells from biolmsbecame visible biogas production stopped. The biogas productiondepends on the substrate available in the uid reactor content. Ifthe substrate in the uid reactor content is depleted biogas pro-duction stagnates. Consequently, the dispersal of cells after 21 daysof anaerobic digestion was most likely related to the substratedepletion.

    In contrast, the biolm formation in reactor H-OL was investi-gated over a period of 7 days. Since biolm carriers were coveredcompletely by a continuous layer of uid reactor content due to itshigh viscosity, the biolm development could only be measured bythe cell numbers. Total cell numbers within the formed biolmswere in the range of 108e109 cells cm2 and doubled within therst 7 days of incubation.

    Biolms that formed in both reactors, H-OL and L-OL, showedEPS formation. The EPS matrix became visible in the form of netlikestructures encompassing single cells attached to biolm carriers

    ation during anaerobic digestion of organic waste, Anaerobe (2013),

  • aeroS. Langer et al. / Anwithin 3 h of anaerobic digestion. These netlike structures becamedenser during fermentation. Independent from the organic loadingrate and cell numbers, biolms were embedded completely in EPSwithin seven days. EPS formation was not dependent on microbialnumbers. Leriche et al. [32] concluded that different organismsproduce differing amounts of EPS and that the amount of EPS in-creases with the age of the biolm. Further, it was remarked thatthe proportion of EPS in mixed biolms did not necessarily reectthe proportions of the microorganisms present, nor did the EPScontribute to the structure and properties of the resulting biolms[33].

    In direct comparison, reactors H-OL and L-OL showed greatdifferences in cell numbers per cm2, biolm formation and biogasproduction. Cell numbers in the uid reactor content of H-OL(3 1010 cells ml1) were three times higher compared to cellnumbers in the uid reactor content of L-OL (11010 cells ml1). Asmentioned before, the initial attachment depends on what landswhere and when [3]. Consequently, biolm formation and struc-ture were dependent on the microorganisms present in the uidreactor contents and substrate characteristics, such as viscosity.Therefore, a high organic loading and high cell numbers in the uidreactor content resulted in the formation of a continuous biolmlayer, whereas a low organic loading and fewer cells in the uidreactor content led to the formation of microcolonies. Furthermore,total cell numbers per cm2 biolm also differed at high (108e

    Fig. 6. CSEM images; formation of the extracellular polymeric substances (EPS) after 1day (1) and 7 days (2). Sample preparation led to artefact formation. The EPS appearedas a netlike structure due to several dehydration steps. Moreover, dehydration ofsamples resulted in deep cracks within the biolms as indicated by the arrow. Scale bar5 mm.

    Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013109 cells cm2) and low organic loadings (107 cells cm2), whereasthe cell density within biolms of both reactors showed similar celldensities in the range of 1011 cells per ml biolm after 7 days ofincubation. The biogas production rate of reactor H-OL wasconsistently higher compared to the biogas production rate of L-OL.This effect was certainly caused by the higher substrate availabilityat a high organic loading rate, but also higher cell numbers con-verting the substrate to biogas might be responsible for this sig-nicant difference in the biogas production rate.

    5. Conclusion

    This investigation underlines the importance of biolms foranaerobic biomass conversion. In biolms the cell densities wereone order of magnitude higher (1011 cells ml1) compared to theuid reactor content (1010 cells ml1), and therefore facilitate theexchange of metabolites in biolms. The biolm formation and itsstructure depended on the organic loading rate, total cell numbers,and substrate availabilities. The biogas production in the performedbatch experiments was strongly correlated to the overall biolmdevelopment. This was shown by the simultaneous detachment ofmicroorganisms within biolms and the decrease of biogas for-mation. The highly dynamic process of anaerobic biolm formationwas described using the parameters cell number and biolmcovered area, whereas the parameters EPS formation and bio-lm thickness were not suitable.

    Biolms of high cell densities can enhance digestion of organicwaste and have positive effects on biogas production. Manybiolm-based reactors are utilising this feature of high reactorloading rates. In biogas practice, biolms attached to the surfaces ofdigested substrates can act in similar manner as in the presentedstudy.

    Acknowledgements

    We thank RMIT Microscopy and Microanalysis Facility (RMIT,Melbourne) and Zentrale Einrichtung fr Mikroskopie (Ulm Uni-versity) for assistance and technical support. The visit abroad(Melbourne, Australia) was funded by DAAD.

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    S. Langer et al. / Anaerobe xxx (2013) 1e88Please cite this article in press as: Langer S, et al., Dynamics of biolm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013ation during anaerobic digestion of organic waste, Anaerobe (2013),

    Dynamics of biofilm formation during anaerobic digestion of organic waste1 Introduction2 Materials and methods2.1 Experimental set up2.2 Fixation of samples2.3 Sample preparation and microscopy2.3.1 Epifluorescence microscopy2.3.2 Light microscopy2.3.3 Conventional scanning electron microscopy2.3.4 Environmental scanning electron microscopy

    2.4 Image analysis

    3 Results3.1 Total cell numbers: fluid reactor contents vs. biofilms3.2 Biofilm surface structure3.3 Biofilm formation and biogas production

    4 Discussion5 ConclusionAcknowledgementsReferences

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