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 . 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 . Biolms are ubiqui-tous in almost every aqueous interface, such as solideliquid or aireliquid interfaces . In most instances where biolms are anuisance, the term microbial fouling or biofouling is widely used. For example, biofouling can be a problem in the food industry, itcontributes to human infections  and it can lead to biocorrosion. 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: email@example.com (S. Langer), firstname.lastname@example.org (D. Schropp), email@example.com (F.R. Bengelsdorf), maazuza.
Contents lists availab
journal homepage: www.else
Anaerobe xxx (2013) firstname.lastname@example.org (M. Othman), email@example.com (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 . 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.
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,
le at ScienceDirect
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 . 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  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 . Due to syntrophic interactions, the pool size of theshuttling intermediate can be kept low, resulting in an efcientcooperation . Further, microorganisms attached to a biolmcarrier form an EPS matrix that offers protection. This EPS matrixprovides mechanical stability and serves as a diffusion barrier .The matrix entraps extracellular enzymes, and prevents the washoff of these enzymes improving the efciency of substrate degra-dation . 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 .
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 . Consequently, substrate composition greatly inuencesthe biodiversity, physiology and structure of biolms . 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 . 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.
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. .
Special biolm carriers made from polypropylene (PP) foi