dynamics of biofilm formation during anaerobic digestion of organic waste

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Anaerobe xxx (2013) 1e8

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Anaerobe

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Molecular biology, genetics and biotechnology

Dynamics of biofilm formation during anaerobic digestionof organic waste

Susanne Langer a,b,*, Daniel Schropp a, Frank R. Bengelsdorf b, Maazuza Othman c,Marian Kazda a

aUlm University, Institute of Systematic Botany and Ecology, Albert-Einstein-Allee 11, 89081 Ulm, GermanybUlm University, Institute of Microbiology and Biotechnology, Albert-Einstein-Allee 11, 89081 Ulm, GermanycRMIT University, Institute of Civil, Environmental and Chemical Engineering, Melbourne, Vic 3001, Australia

a r t i c l e i n f o

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

Keywords:Anaerobic biofilmBiofilm formationAnaerobic digestionBiogas

* Corresponding author. Ulm University, Institute onology, Albert-Einstein-Allee 11, 89081 Ulm, Germany

E-mail addresses: [email protected] (S.ulm.de (D. Schropp), [email protected]@rmit.edu.au (M. Othman), marian.kazda@un

1075-9964/$ 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.2013.11

a b s t r a c t

Biofilm-based reactors are effectively used for wastewater treatment but are not common in biogasproduction. This study investigated biofilm dynamics on biofilm carriers incubated in batch biogas re-actors at high and low organic loading rates for sludge from meat industry dissolved air flotation units.Biofilm formation and dynamics were studied using various microscopic techniques. Resulting micro-graphs were analysed for total cell numbers, thickness of biofilms, biofilm-covered surface area, and thearea covered by extracellular polymeric substances (EPS).

Cell numbers within biofilms (1011 cells ml�1) were up to one order of magnitude higher compared tothe numbers of cells in the fluid reactor content. Further, biofilm formation and structure mainlycorrelated with the numbers of microorganisms present in the fluid reactor content and the organicloading. At high organic loading (45 kg VS m�3), the thickness of the continuous biofilm 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 m�3), only microcolonies were detectable. Those microcolonies increased in their frequency ofoccurrence during ongoing fermentation. Independently from the organic loading rate, biofilms wereembedded completely in EPS within seven days. The maturation and maintenance of biofilms changedduring the batch fermentation due to decreasing substrate availability. Concomitant, detachment ofmicroorganisms within biofilms was observed simultaneously with the decrease of biogas formation.

This study demonstrates that biofilms of high cell densities can enhance digestion of organic waste andhave positive effects on biogas production.

� 2013 Elsevier Ltd. All rights reserved.

1. 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 efficiency of the biogas processcan be further improved by engineering the microbial community.

A possible approach to improve the biogas process is the addi-tion of biofilm carriers (e.g. plant material) to the biogas reactors.Bacteria and archaea involved in the methane production duringanaerobic digestion could attach to biofilm carriers and form

f Microbiology and Biotech-. Tel.: þ49 731 5022713.Langer), daniel.schropp@uni-(F.R. Bengelsdorf), maazuza.i-ulm.de (M. Kazda).

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biofilms. Biofilms are assemblages of microorganisms, attached to asurface and encased in an extracellular polymeric substances (EPS)matrix, that functions as a cooperative consortium [2]. The struc-ture of microbial communities ranges frommonolayers of scatteredsingle cells to thick, mucous structures of macroscopic dimensions[3].

The biofilm life cycle can be divided into three stages: theattachment of single cells to a surface, the maturation of the biofilmto complex microcolonies and the cell dispersal of highly motileplanktonic cells. The biofilm mode of life is a feature common tomost microorganisms in natural habitats [2]. Biofilms are ubiqui-tous in almost every aqueous interface, such as solideliquid or aireliquid interfaces [4]. In most instances where biofilms 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, biofilms do not only reveal negative effects. The

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

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S. Langer et al. / Anaerobe xxx (2013) 1e82

application of biofilms 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, biofilms playan important role as they create the basis of diverse aerobic andanaerobic reactors [8]. Biofilms contribute to a more efficientdegradation of organic substrates and to a higher biogas ormethane yield. Moreover, biofilm formation can result in a morestable degradation process. There are several explanations for thesepositive effects of biofilms on anaerobic digestion. Microorganismsattach to surfaces and build up complex aggregates. Thereby, thebiomass increases, due to higher cell densities within the biofilms.Thus, more efficient degradation of organic substrates is shown[9,10]. For instance, Zak [9] demonstrated that the addition of aplant-based biofilm carrier improves biogas formation. The specificmethane yield and the organic drymatter degradation increased byup to 7% and 10%, respectively, due to the microbial biomass on thebiofilm carriers.

The biofilm mode of life offers advantages like syntrophic in-teractions due to the physical vicinity of microorganisms withinbiofilms. 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 efficientcooperation [12]. Further, microorganisms attached to a biofilmcarrier 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 efficiency of substrate degra-dation [14]. The diffusion barrier also prevents the entry of harmingsubstances into the biofilm. Thus, cells within biofilms 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 biofilmformation in respect to different organic loading rates. Therefore,biogas reactors with low and high organic loading rates were setup. Biofilm carriers were incubated in these biogas reactors andremoved after certain periods to investigate the biofilms. Moreover,cell numbers within the formed biofilms and fluid reactor contentsof the biogas reactors were quantified. The formation of biofilms isinfluenced by different factors like genotypic and physico-chemicalfactors [3]. Consequently, substrate composition greatly influencesthe biodiversity, physiology and structure of biofilms [18]. Thus, thebiofilm structures were also investigated in respect to differentorganic loading rates. Moreover, biogas production of biogas re-actors were measured and compared with biofilm formation anddevelopment of cell numbers within biofilms and fluid 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 flotation(DAF) sludge collected from slaughterhouse wastewater (UlmerFleisch GmbH, Ulm, Germany). The organic loading amounted to

Please cite this article in press as: Langer S, et al., Dynamics of biofilm formhttp://dx.doi.org/10.1016/j.anaerobe.2013.11.013

45 kg VS m�3 (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 m�3. 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 Procházka et al. [21].

Special biofilm carriers made from polypropylene (PP) foil wereused for microscopical analysis of biofilm characteristics. PP-discs(Ø 9 mm) were punched out of a polypropylene foil (Ø 0.5 mm)and a hole was made in the middle of each PP-disc to slide severalPP-discs on a stainless steel wire (Ø 1 mm) with a length of 20 cm.One end of the wirewas formed to a loop in order to fix a nylon lineto hang the biofilm 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 biofilm 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 fluidreactor contents (frc) were fixed 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 fixation 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.

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

2.3. Sample preparation and microscopy

2.3.1. Epifluorescence microscopyTotal cell numbers of microorganisms in frc and in biofilms

attached to PP-discs were analysed by epifluorescence microscopy.Therefore, fixed 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 (Ø 0.1 mm).20 ml of the homogeneous cell suspension was dropped onto eachwell of a Teflon-coated slide (8 wells, Ø 6 mm; Menzel GmbH & Co.KG, Germany) and dried for 15 min at 60 �C. In order to fix cells, theslidewas pulled through the flame 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, flushed with cold double-distilled H2O, andimmediately dried with compressed air. Before microscopy, twodrops of Citifluor� AF1 (Citifluore Ltd., UK) were applied to the


Table 1Total cell numbers per ml fluid reactor content from lab-scale biogas reactors set upwith high (H-OL, 45 kg VS m�3) and low organic loadings (L-OL, 15 kg VS m�3).

Time [d] H-OL L-OL

Cells ml�1 fluid reactor content0 (4.42 � 1.93) � 1010 (na ¼ 30) (1.20 � 0.32) � 1010 (n ¼ 10)7 (2.64 � 0.51) � 1010 (n ¼ 30) (1.07 � 0.63) � 1010 (n ¼ 10)14 (3.38 � 1.27) � 1010 (n ¼ 30) (1.01 � 0.27) � 1010 (n ¼ 10)21 (2.09 � 0.54) � 1010 (n ¼ 30) (0.76 � 0.23) � 1010 (n ¼ 10)28 (2.43 � 0.72) � 1010 (n ¼ 30) (0.61 � 0.20) � 1010 (n ¼ 10)Cells ml�1 biofilm7 (2.96 � 1.05) � 1011 (n ¼ 20) (2.09 � 0.99) � 1011 (n ¼ 10)

a n ¼ number of analysed epifluorescence images.

S. Langer et al. / Anaerobe xxx (2013) 1e8 3

slide, and a cover slip was positioned to cover all wells. The fluo-rescence signals of samples from reactor H-OLwere detected by theLeitz DMRBE epifluorescence microscope (Leica MicrosystemsGmbH, Germany) and fluorescence signals of stained samples fromreactor L-OL was detected by the Leica DM 2500. Filter I3 (excita-tion filter: 450e490 nm, dichromatic mirror: 510 nm, suppressionfilter: 515 nm) was used for both microscopes to detect SYBR� Goldstained microorganisms. Epifluorescence 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. Epifluorescence 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 biofilms, PP-discs with attached

biomass were fixed and crosscut. After fixation, PP-discs withattached biofilms were washed three times for 5e10 min in PBS. Asecond fixationwas 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 fixed 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 biofilm 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 biofilms. After fixation of biofilmsattached 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-films 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 2�10�7 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 biofilms sampled from reactor L-OL. For visualisationof biofilms 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 epifluorescence images were auto-matically enumerated by the software LAS Image Analysis a part ofthe Leica Application Suite V3 (Leica Microsystems GmbH, Ger-many) and Mac Biophotonics ImageJ (Wayne Rasband, Freeware).


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

3. Results

3.1. Total cell numbers: fluid reactor contents vs. biofilms

Total cell numbers in the fluid reactor contents of lab-scalebiogas reactors with a high (H-OL, 45 kg VS m�3) and a low (L-OL, 15 kg VS m�3) organic loading were estimated during anaerobicdigestion over a period of 28 days (Table 1).

In the fluid reactor content of the biogas reactor with a highorganic loading 3 � 1010 cells ml�1 were found in average. Cellnumbers in the fluid 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 ml�1

fluid reactor content, two-thirds less thanthe cells in reactor H-OL. Moreover, cell numbers in the fluid 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 biofilms formed during the incubation ofbiofilm carriers in reactor H-OL or L-OL were estimated after sevendays of anaerobic digestion. At the same time point, samples fromthe fluid reactor contents were collected and the cell numbers wereestimated by epifluorescence microscopy. The cell numbers withinthe biofilms and the fluid reactor contents were compared to eachother (Table 1). Total cell numbers within the biofilms(1011 cells ml�1) were one order of magnitude higher compared tothe numbers of cells in the fluid reactor content (1010 cells ml�1).Additionally, cell numbers within the biofilms of reactor H-OL wereslightly higher compared to cell numbers within biofilms of reactorL-OL.

3.2. Biofilm surface structure

The structure of biofilms can be influenced bymany factors. Oneof these factors can be the amount of the available substrate. Highorganic loading rates contributed to a continuous biofilm layer. Athin layer of reactor content immediately covered biofilm 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


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


knobbly structures. In particular, semi-spherical microcoloniesappeared to be fluffy on the surface.

3.3. Biofilm formation and biogas production

The formation of biofilms is a dynamic process that depends onmany different factors. The thickness of biofilms 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 biofilms appeared in the form of a continuouslayer. During microscopy it became obvious that plant and otherunidentified particles within the fluid reactor content of the biogasreactor mainly contributed to the thickness of the biofilm layer (Fig2). Although, the biofilm layer was continuous it showed greatsurface irregularities. Consequently, there was no remarkable in-crease of the biofilm thickness noticed within the observation timeof seven days. Moreover, the measured values of the biofilmthickness showed great variations, also within samples collected atthe same time point (Fig 3). The thickness of the biofilms 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 significant increase in biofilm thicknessmeasurable during the time of observation.

At low organic loadings microcolonies were formed instead of acontinuous biofilm layer. These microcolonies were not detectableafter one day (Fig 4.1), but were first 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 biofilm-covered surface area andtotal cell numbers per cm�2 biofilm (Fig 5.1) were investigated for a

Fig. 2. Light microscopic images; semi cross section


period of 28 days. Since at low organic loadings, the biofilm carrierwas not completely covered by a biofilm layer, changes in thebiofilm growth were measurable by the biofilm-covered surfacearea. After one day of incubation, only 14% of the biofilm carrier wascovered by biofilm. The biofilm covered surface area increased up to51% after 21 days of incubation and dropped to 13% after 28 days.

Total cell numbers per cm2 biofilm carrier showed a similardevelopment. After one day of incubation at low organic loadings,1.74 � 107 cells cm�2 were detectable. In the first seven days ofanaerobic digestion cell numbers per cm2 biofilm doubled to3.55 � 107 cells cm�2 and stayed stable until day 21 of anaerobicdigestion. After 21 days, cell numbers dropped to1.17� 107 cells cm�2. In the period of 21 days, when biofilm growthwere detectable in respect to microcolony formation, biofilmcovered surface area and cell numbers per cm2 biofilm carrier, anincrease in biogas production was measured. Interestingly, at thesame time, biofilm formation decreased and cell dispersal becamevisible in the form of hollow microcolonies the biogas productiondecreased as well. Statistical analysis showed a strong correlationbetween biofilm development and biogas production at loworganic loading rates (Spearman R ¼ 0.8, N ¼ 5, r < 0.01).

At a high organic loading the biofilm covered surface area andtotal cell numbers per cm2 biofilm (Fig 5.2) were investigated for atime period of seven days. The biofilm carrier was coveredcompletely by the reactor content, due to its high viscosity at highorganic loading rates. Consequently, changes in biofilm growthwere not measurable by the biofilm covered surface area. Thebiofilm covered surface area was 100% during the whole observa-tion period of seven days. Total cell numbers per cm�2 biofilmcarrier increased during anaerobic digestion. After one day of in-cubation, 0.76� 108 cells cm�2 biofilmwere detectable. After sevendays of anaerobic digestion, the cell numbers doubled to1.5 � 109 cells cm�2 biofilm similar to cell numbers at low organicloading rates. Since the biofilm formation was investigated only

s of a biofilm with incorporated plant particles.


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


over a period of 7 days no connection of biofilm formation to thebiogas production could be made (Fig 5.4).

Microorganisms within biofilms were embedded in an EPSmatrix that consisted mainly of polysaccharides. In ESEM images ofbiofilms, 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 ofincubation (Fig 6.1) and increased to an almost continuous layerduring further anaerobic digestion independently from the organicloading (Fig 6.2).

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

4. Discussion

The dynamics of biofilm formation were investigated duringanaerobic digestion. Total cell numbers within biofilms and fluidreactor contents of the biogas reactors with different loading rateswere determined. In general, total cell numbers in the fluid reactorcontents of the experimental reactors fed with DAF sludge were inthe range of 1010 cells ml�1. Similar results were previously re-ported by Bengelsdorf et al. [24] and Krakat et al. [25]. In contrast,cell numbers within biofilms were one order of magnitude higherin the range of 1011 cells ml�1. Most likely the mentioned positiveeffects of biofilms on the biogas process resulted from the high celldensity and physical vicinity within biofilms, 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 biofilm formation, might explain this slightdifference.

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

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

Fig. 5. Biofilm formation during anaerobic digestion at low (1, 3) and high (2, 4) organic loading rates compared to total cell numbers within the fluid reactor content and the biogasproduction of the biogas reactors. The results were displayed over a period of 28 days and 7 days, respectively. ( ) Biofilm covered surface area [%], ( ) cells per cm2 biofilm[cells cm�2], ( ) cells per ml biofilm [cells ml�1], ( ) cells per ml fluid reactor content [cells ml�1], ( ) biogas production rate [Nl kg�1 VS�1 d�1].


continuous biofilm layer. In contrast, lower organic loadingsresulted in microcolony formation. This type of biofilm 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 biofilm formation,since the microorganisms have a marked effect on the structure[26].

Although a continuous biofilm layer was formed at high organicloadings, biofilms showed irregularities. As a consequence, thethickness of the biofilms 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 biofilms are highly het-erogeneous structures. These irregularities in the structure of bio-films are not only characteristic for multispecies biofilms, but alsofor pure culture biofilms [26]. Further Walker et al. [27] described abiofilm as a non-uniform structure with a variable thickness thatcan change significantly over distances of 10 mm or less [28].Therefore, the thickness of biofilms was not a suitable parameter toanalyse biofilm formation during anaerobic digestion. One of themodel organisms for biofilm development is Pseudomonas aerugi-nosa. In comparison to the results of this study, other researcherscharacterised the variability in thickness of P. aeruginosa biofilms.For example, the mean of the thickness of a biofilm formed byP. aeruginosa was 33 mm, with a range of 13.3e60 mm [29].

The dynamics of biofilms were analysed in terms microcolonyformation, biofilm covered surface area and total cell numbers inrespect to different organic loading rates. The biofilm 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 biofilm covered surface area and cell numbers per cm2

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

were in the range of 107e108 cells cm�2.Within the following 14 days of fermentation cell numbers per

cm2 biofilm carrier and the biofilm 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 biofilms indi-cated by reduced growth rates and development of unique


structures [26]. The biofilm mode of life is restricted for bacterialgrowth and the transcriptome of mature biofilm, on average, re-sembles that of stationary-phase cells [30]. The last stage in thedevelopment of biofilms is the detachment and dispersal of mi-croorganisms. Microorganisms break the ‘biofilm 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 biofilms undergo internal disintegration, leaving behind“hollow” shell-like structures [31]. Furthermore, cell number percm2 and the biofilm covered area decreased. The dispersal of mi-croorganisms from biofilms can be influenced by many factors,especially alterations in the habitat and environmental quality,such 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 biofilm formation the biogas production ofbiogas reactors was analysed during anaerobic digestion and linkedto the biofilm development. At low organic loadings biofilmdevelopment was correlated to biogas production. Biogas accu-mulated within the first 21 days of anaerobic digestion anddecreased afterwards. When dispersal of cells from biofilmsbecame visible biogas production stopped. The biogas productiondepends on the substrate available in the fluid reactor content. Ifthe substrate in the fluid 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 biofilm formation in reactor H-OL was investi-gated over a period of 7 days. Since biofilm carriers were coveredcompletely by a continuous layer of fluid reactor content due to itshigh viscosity, the biofilm development could only be measured bythe cell numbers. Total cell numbers within the formed biofilmswere in the range of 108e109 cells cm�2 and doubled within thefirst 7 days of incubation.

Biofilms 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 biofilm carriers


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 biofilms as indicated by the arrow. Scale bar5 mm.


within 3 h of anaerobic digestion. These netlike structures becamedenser during fermentation. Independent from the organic loadingrate and cell numbers, biofilms 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 biofilm. Further, it was remarked thatthe proportion of EPS in mixed biofilms did not necessarily reflectthe proportions of the microorganisms present, nor did the EPScontribute to the structure and properties of the resulting biofilms[33].

In direct comparison, reactors H-OL and L-OL showed greatdifferences in cell numbers per cm2, biofilm formation and biogasproduction. Cell numbers in the fluid reactor content of H-OL(3 � 1010 cells ml�1) were three times higher compared to cellnumbers in the fluid reactor content of L-OL (1�1010 cells ml�1). Asmentioned before, the initial attachment depends on ‘what landswhere and when’ [3]. Consequently, biofilm formation and struc-ture were dependent on the microorganisms present in the fluidreactor contents and substrate characteristics, such as viscosity.Therefore, a high organic loading and high cell numbers in the fluidreactor content resulted in the formation of a continuous biofilmlayer, whereas a low organic loading and fewer cells in the fluidreactor content led to the formation of microcolonies. Furthermore,total cell numbers per cm2 biofilm also differed at high (108e


109 cells cm�2) and low organic loadings (107 cells cm�2), whereasthe cell density within biofilms of both reactors showed similar celldensities in the range of 1011 cells per ml biofilm 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-nificant difference in the biogas production rate.

5. Conclusion

This investigation underlines the importance of biofilms foranaerobic biomass conversion. In biofilms the cell densities wereone order of magnitude higher (1011 cells ml�1) compared to thefluid reactor content (1010 cells ml�1), and therefore facilitate theexchange of metabolites in biofilms. The biofilm 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 biofilmdevelopment. This was shown by the simultaneous detachment ofmicroorganisms within biofilms and the decrease of biogas for-mation. The highly dynamic process of anaerobic biofilm formationwas described using the parameters “cell number” and “biofilmcovered area”, whereas the parameters “EPS formation” and “bio-film thickness” were not suitable.

Biofilms of high cell densities can enhance digestion of organicwaste and have positive effects on biogas production. Manybiofilm-based reactors are utilising this feature of high reactorloading rates. In biogas practice, biofilms 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 für Mikroskopie (Ulm Uni-versity) for assistance and technical support. The visit abroad(Melbourne, Australia) was funded by DAAD.

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dynamics of biofilm formation during anaerobic digestion of organic waste

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