Exploring the Mechanism of Biocatalyst Inhibition in Microbial Desulfurization

Abstract

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 M with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC 50 s) for DszA, DszB, and DszC were measured to be 60 ؎ 5 M, 110 ؎ 10 M, and 50 ؎ 5 M, respectively. The fact that the IC 50 s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation. Biodesulfurization (BDS) is a process in which microorganisms, typically, bacteria, are used to reduce the level of sulfur in fuels derived from crude oil, including diesel and gasoline. Over the last 20 years, interest in BDS as an alternative to hydrodesulfurization (HDS), which is the current desulfurization standard in the oil industry, has increased. HDS uses a metal catalyst along with hydrogen gas (H 2 ) at high temperature and pressure to remove sulfur from organic sulfur compounds and generate H 2 S gas (1). Major drawbacks of HDS include steric hindrance of the metal catalysts by certain recalcitrant compounds and large energy consumption due to process operation at high temperature and pressure (1). Recalcitrant compounds include the parent molecule dibenzothiophene (DBT) and some of its alkylated derivatives, such as 4-methyldibenzothiophene (4-DBT) and 4,6-dimethyldibenzothiophene (4,6-DBT). BDS can potentially be used to remove the sulfur that cannot be removed by HDS, though it is likely not a replacement for the current HDS infrastructure. The use of more than one desulfurization technology may be necessary to meet the increasingly stringent sulfur regulations (1).There is a wide range of microorganisms known to have BDS capability (2). Such microorganisms typically desulfurize DBT by one of two pathways: the Kodama pathway or the 4S pathway (1). The Kodama pathway i...

Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds

“…As shown in figure 4A, a high initial removal rate of DBT was observed, but the reaction slowed down after 15 min and 30% of the substrate (DBT) remained in the medium at the end of the resting cell process. These results, therefore, confirmed in vivo the inhibitory effects of HBPS on the DszC activity observed in vitro (Abin-Fuentes et al, 2013).The dszA1-D1 cassette (Fig. 1C) completely converted DBTO 2 into HBPS in about 120 min (Fig.…”

“…Moreover, decay of the BDS activity due to the accumulation of the sulfur-free end product 2HBP in bacterial biocatalysts has been reported (Abin-Fuentes et al, 2013). It has been shown that bacteria harboring identical dsz genes provide different BDS rates due to the influence of the host metabolism (Kilbane, 2006 (Kilbane, 2006;Li et al, 2008;Ma et al, 2006;Shavandi et al, 2009; Wang et al, 2011).…”

“…Despite all these successful trials to produce genetically improved biocatalysts, so far, none of the deployed gene manipulation techniques has resulted in a BDS rate meeting the industrial requirement for the development of a commercial BDS process (Boniek et al, 2015;Kilbane, 2006). Moreover, no real efforts have been taken to solve HBPS and 2HBP-related feedback inhibition of the BDS process (Abin-Fuentes et al, 2013). Since the 4S pathway consists of three serial and irreversible functional modules (Fig.…”

“…3A) than with a biocatalyst expressing the complete dszB1A1C1-D1 module ( Fig. 2A), pointing out the inhibitory effect of 4S-pathway compounds on the activity of DszC monooxygenase (Abin-Fuentes et al, 2013). To study further this inhibitory effect in vivo, we monitored the conversion of DBT into HBPS by using the dszA1C1-D1 cassette (Fig.…”

“…Hence, to increase the 16 bioconversion of DBT into 2HBP we designed an artificial 4S pathway based on the construction of synthetic bacterial consortia. This approach of sequential desulfurization using segregated steps of the 4S pathway should also avoid the inhibitory effect that HBPS and 2HBP exert on DszC in monocultures (Abin-Fuentes et al, 2013).A first bacterial consortium was made of P. putida KT2440 (pIZdszC1-D1) cells, which convert DBT into DBTO 2 (Fig. 3A), and P. putida KT2440 (pIZdszB1A1-D1) cells, which convert DBTO 2 into 2HBP (Fig.…”