Bioresource Technology 155 (2014) 50–56 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Cellulose fermentation by Clostridium thermocellum and a mixed consortium in an automated repetitive batch reactor ⇑ Parker T. Reed, Javier A. Izquierdo 1, Lee R. Lynd Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States highlights An automated repetitive batch fermentation system was developed. A strong correlation was observed between CO2 produced and cellulose consumed. C. thermocellum and a mixed consortium exhibit accelerated substrate utilization. Similar numbers of cycles were needed to reach maximum CO2 production rates. Maximum CO2 production rates and cycle times were very similar for both cultures. article info abstract Article history: An automated repetitive batch fermentation system was developed to facilitate the study of microbial Received 4 October 2013 cellulose utilization. The system was operated with Avicel as the carbon source and either Clostridium Received in revised form 10 December 2013 thermocellum ATCC 27405 or a consortium enriched from compost as inocula. Multiple cycles of growth Accepted 12 December 2013 medium addition, incubation, and medium removal were performed with each inoculum. Removal and Available online 25 December 2013 addition of media were automatically initiated when CO2 production fell to 90% of the cycle’s peak. A strong correlation was observed between CO2 production and cellulose consumption, suggesting that Keywords: the online signal was a good proxy for substrate utilization. Both cultures exhibited accelerated substrate Automated batch fermentation utilization and a decrease in cycle time. About the same number of cycles was required to reach maxi- Cellulose degradation Enrichment cultures mum CO2 production for both cultures. Notably, the magnitudes of the maximum CO2 production rate C. thermocellum and cycle times were very similar for both C. thermocellum in pure culture and an environmental consortium. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction complex called the cellulosome (Shoham et al., 1999; Gilbert, 2007). The structure of the cellulosome is believed to foster syner- Cellulolytic microorganisms play key roles in the global carbon gistic interactions between enzyme components, and there is evi- cycle and are of significant interest for the production of renewable dence that cellulosomes are several-fold more effective when fuels and chemicals (Lynd et al., 2002; Himmel et al., 2007). they act as part of cellulose–enzyme–microbe complexes as com- Microbes which rely on non-oxidative processes for provision of pared to when they act without a physical association with cells metabolically useful energy are of particular interest in the latter (Lu et al., 2006). On the other hand, enrichment cultures derived context, since a very high fraction of the substrate enthalpy is from inocula taken from nature are also useful in the discovery conserved in products (Lynd et al., 2005). of novel species and cell functions, and in gaining valuable insights Pure culture studies play a key role in understanding microbial into the mechanics of lignocellulose degradation (Haruta et al., cellulosic utilization. Clostridium thermocellum is a widely-studied 2002; Burrell et al., 2004; Izquierdo et al., 2010; Carver et al., cellulolytic, thermophilic anaerobic bacterium, which solubilizes 2011; De Angelis et al., 2012; Ronan et al., 2013). In particular, cellulose primarily via a cell surface-associated multi-enzyme studies using enrichment cultures allow evaluation of lignocellu- lose solubilization under conditions that more nearly approximate those present in nature as compared to pure cultures. ⇑ Corresponding author at: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States. Tel.: +1 (603) 646 2231; fax: +1 (603) 646 2277. Although batch and continuous cultures each have merits for E-mail address: [email protected] (L.R. Lynd). characterizing microbial solubilization of lignocellulose, batch cul- 1 Current address: Center for Agricultural and Environmental Biotechnology, RTI tures are of interest because cultures grow at their maximum International, Research Triangle Park, NC 27709, United States. 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.051 P.T. Reed et al. / Bioresource Technology 155 (2014) 50–56 51 growth rate, often achieve a higher fractional substrate utilization 2.3. Automated repetitive batch fermentation system (Wisselink et al., 2007), can avoid metabolic responses associated with stationary phase (Huang et al., 2008), and for some purposes A system was developed to conduct repetitive batch fermenta- are more time-efficient to study. On the other hand, continuous tion experiments with the benefits of online measurements and cultures allow for cultures to evolve, and in the case of mixed automatic batch transfers. For each batch transfer, a well-mixed enrichment adjust their species composition. Automated repeated reactor was drained to a specified liquid level of 15% or 150 mL batch (ARB) culture, in which a fraction of the cell broth is used as of the original 1 L and refilled to the original volume of 1 L with an inoculum for successive batch cycles, combines some of the 850 mL of fresh media. This allowed the cells that remained in merits of conventional batch and continuous cultures. ARB cultures the vessel to serve as the inoculum for the next transfer. The move- have been used to enrich and characterize microbes and microbial ment of liquid from one vessel to another was achieved by pressur- communities on a variety of substrates (Wisselink et al., 2007; ization of the donor vessel, while the recipient vessel was kept at Huang et al., 2008; Tolvanen et al., 2011), although not for cellu- slightly higher than atmospheric pressure. losic substrates. The system studied consisted of a 1 L water-jacket-heated Here, an ARB system was developed for the cultivation of Sartorius Biostat A fermentor (Sartorius Stedim, Gottingen, thermophilic, cellulolytic bacteria on a model cellulosic substrate Germany), a large 10 or 20 L media carboy with a magnetic stir (Avicel), observe the behavior of pure cultures of C. thermocellum plate, a second 1 L water-jacket-heated vessel used as a media in this system, and compare the performance of mixed consortia preheater, an automated sampler system, a waste carboy, and obtained from an environmental inoculum. an off gas CO2 analyzer (Fig. 1). A control program was created in LabVIEW (National Instruments, Austin, TX) to control the functions of the automated repetitive batch system and aid the 2. Methods user in programming experiments. The system used an electro- magnetic relay board controlled by LabVIEW (Table 1). This relay 2.1. Inocula board controlled 11 solenoid pinch valves, the liquid level sen- sors, fraction collector, and sample pump, as depicted in Fig. 1 C. thermocellum ATCC 27405 was obtained from the American and Table 1. All connections in the system were made with size Type Culture Collection (Manassas, Virginia), maintained on MTC 16 Norprene tubing. medium, and stored at À80 °C until grown in a reactor containing An electronic liquid level sensor was designed to accurately 1 L of low carbon (LC) medium with 5 g/L Avicel PH-105 to 90% of measure the two volumes of liquid, 150 mL and 850 mL, required peak CO and stored in 50 mL aliquots in purged serum bottles at 2 for the batch transfer process. The liquid level system (Fig. 1) con- À80 °C until needed. Bottles were thawed at room temperature sisted of a stainless steel probe electrically isolated from the reac- with no agitation and injected directly into the reactor for tor headplate. inoculation. The automated sampler module consisted of 3 normally closed A cellulolytic consortium was obtained after three transfers pinch valves, a port that reached the bottom of the reactor, a N2 gas from a primary enrichment using thermophilic horse manure com- line, a peristaltic pump, and a BioFrac fraction collector (Bio-Rad, post from the Dartmouth College composting facility, as previously Hercules, CA). The fraction collector and the pump were both con- described (Izquierdo et al., 2010), and designated DC3-3. These trolled using LabVIEW by relays in an USB-IIRO 16 relay board. The transfers were grown in Defined Minimal Media (DMM) with fraction collector featured a robotic arm and 15 mL conical tubes in 3 g/L Avicel PH-105 in 125 mL serum bottles. The inoculum for fer- a water bath that was cooled to 1 °C by an immersion coil fed by an mentation was prepared through an additional transfer in Low Car- external chiller using propylene glycol. The sample port and all bon (LC) media with 5 g/L Avicel PH-105 then grown in a 1 L tubes were purged at the end of each sampling event. 22 mL sam- fermentor on LC media with 5 g/L Avicel PH-105. 50 mL aliquots ples were taken at the beginning and end of each batch cycle as were taken at 90% of peak CO and stored in purged serum bottles 2 well as every 2 h during the cycle. at À80 °C until needed. Bottles were thawed at room temperature with no agitation and injected directly into the reactor to inoculate. 2.4. System operation 2.2. Media formulation Each repetitive batch experiment consisted of an initial fermen- Low carbon (LC) medium, used for repetitive batch experi- tation and then 14 subsequent transfers for a total of 15 fermenta- ments, was previously developed (Holwerda et al., 2012) to reduce tion cycles. Growth media was prepared in one 15 L batch in a 20 L the amount of carbon other than the primary growth substrate and carboy and used for all of the cycles of a given experiment to main- to increase the accuracy of analytical techniques such as total or- tain consistency.