The potential of osmolytes and their precursors to alleviate osmotic stress of anaerobic granular sludge.

Increasing amounts of saline (waste)water with high concentrations of organic pollutants are generated globally. In the anaerobic (waste)water treatment domain, high salt concentrations are repeatedly reported to inhibit methanogenic activity and strategies to overcome this toxicity are needed. Current research focuses on the use of potential osmolyte precursor compounds for osmotic stress alleviation in granular anaerobic sludges upon exposure to hypersalinity shocks. Glutamic acid, aspartic acid, lysine, potassium, gelatine, and tryptone were tested for their potential to alleviate osmotic stress in laboratory grown and full - scale granular sludge. The laboratory grown granular sludge was adapted to 5 (R5) and 20 (R20) g Na+/L. Full-scale granular sludge was obtained from internal circulation reactors treating tannery (waste)water with influent conductivity of 29.2 (Do) and 14.1 (Li) mS/cm. In batch experiments which focused on specific methanogenic activity (SMA), R5 granular sludge was exposed to a hypersalinity shock of 20 g Na+/L. The granular sludge of Do and Li was exposed to a hypersalinity shock of 10 g Na+/L with sodium acetate as the sole carbon source. The effects on R20 granular sludge were studied at the salinity level to which the sludge was already adapted, namely 20 g Na+/L. Dosing of glutamic acid, aspartic acid, gelatine, and tryptone resulted in increased SMA compared to only acetate fed batches. In batches with added glutamic acid, the SMA increased by 115% (Li), 35% (Do) and 9% (R20). With added aspartic acid, SMA increased by 72% (Li), 26% (Do), 12% (R5) and 7% (R20). The addition of tryptone resulted in SMA increases of 36% (R5), 17% (R20), 179% (Li), and 48% (Do), whereas added gelatine increased the SMA by 30% (R5), 14% (R20), 23% (Li), and 13% (Do). The addition of lysine, meanwhile, gave negative effects on SMA of all tested granular sludges. Potassium at sea water Na/K ratio (27.8 w/w) had a slight positive effect on SMA of Do (7.3%) and Li (10.1%), whereas at double the sea water ratio (13.9% w/w) had no pronounced positive effect. R20 granular sludge was also exposed to hyposalinity shock from 20 down to 5 g Na+/L. Glutamate and N-acetyl-ß-lysine were excreted by microbial consortium in anaerobic granular sludge adapted to 20 g Na+/L upon this exposure to hyposalinity. A potential consequence when applying these results is that saline streams containing specific and hydrolysable proteins can be anaerobically treated without additional dosing of osmolytes.

Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.