Reduction of the hydraulic retention time at constant high organic loading rate to reach the microbial limits of anaerobic digestion in various reactor systems Ayrat M. Ziganshin a , Thomas Schmidt b,c , Zuopeng Lv d , Jan Liebetrau b , Hans Hermann Richnow e , Sabine Kleinsteuber d , Marcell Nikolausz d,⇑ a Department of Microbiology, Kazan (Volga Region) Federal University, Kazan 420008, The Republic of Tatarstan, Russia b Department of Biochemical Conversion, Deutsches Biomasseforschungszentrum gGmbH, 04347 Leipzig, Germany c Faculty of Agricultural and Environmental Sciences, University of Rostock, 18059 Rostock, Germany d Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany e Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany highlights  Anaerobic digestion at extreme short HRT was investigated in various reactors.  Functional markers for H 2 and CH 4 producing pathways were analyzed on mRNA level.  Stable isotope analysis of the biogas was applied to assess methanogenic pathways.  Depending on HRT Clostridiales and Spirochaetales were the most active in reactors.  Increased activity of Methanosaeta was found during HRT decrease in CSTR and ASBR. article info Article history: Received 6 December 2015 Received in revised form 22 January 2016 Accepted 25 January 2016 Available online xxxx Keywords: Biogas Thin stillage Fe–Fe-hydrogenases Methanogenic activity Stable isotope fingerprinting abstract The effects of hydraulic retention time (HRT) reduction at constant high organic loading rate on the activ- ity of hydrogen-producing bacteria and methanogens were investigated in reactors digesting thin stillage. Stable isotope fingerprinting was additionally applied to assess methanogenic pathways. Based on hydA gene transcripts, Clostridiales was the most active hydrogen-producing order in continuous stirred tank reactor (CSTR), fixed-bed reactor (FBR) and anaerobic sequencing batch reactor (ASBR), but shorter HRT stimulated the activity of Spirochaetales. Further decreasing HRT diminished Spirochaetales activity in systems with biomass retention. Based on mcrA gene transcripts, Methanoculleus and Methanosarcina were the predominantly active in CSTR and ASBR, whereas Methanosaeta and Methanospirillum activity was more significant in stably performing FBR. Isotope values indicated the predominance of aceticlastic pathway in FBR. Interestingly, an increased activity of Methanosaeta was observed during shortening HRT in CSTR and ASBR despite high organic acids concentrations, what was supported by stable isotope data. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction One of the rapidly advancing areas of modern biotechnology is the development of sustainable waste treatment technologies. Much attention is paid to the efficient anaerobic digestion of organic substances, including agricultural, industrial, and house- hold wastes, with the production and utilization of energy rich bio- gas. Anaerobic conversion of waste organic matter into biogas in engineered systems is also a promising way of reducing uncontrolled greenhouse gas emissions. However, it is also recommended to improve the digestate storage to reduce further methane emissions (Mata-Alvarez et al., 2000; Holm-Nielsen et al., 2009). Among the main operational conditions that need to be period- ically monitored in anaerobic digesters, hydraulic retention time (HRT) is one of the key process parameters. Assuming volume con- stancy this parameter is calculated from the working volume of the reactor that is divided by the daily feeding volume of the substrate; therefore, it is closely linked to the organic loading rate (OLR) and defines the average residence time of the substrate in the system. Current biogas plants in the agricultural sector usually apply long http://dx.doi.org/10.1016/j.biortech.2016.01.096 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +49 341 2434 566. E-mail address: marcell.nikolausz@ufz.de (M. Nikolausz). Bioresource Technology xxx (2016) xxx–xxx Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Please cite this article in press as: Ziganshin, A.M., et al. Reduction of the hydraulic retention time at constant high organic loading rate to reach the micro- bial limits of anaerobic digestion in various reactor systems. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.096