Exposure to high salinity stimulated engineered S. elongatus to produce isobutanol
To investigate how to improve isobutanol production of engineered cyanobacteria using environmental stress, a plasmid pJW12 containing the genes kivD and adhA from L. lactis was constructed under the control of the promoter Ptrc, the gene alsS from B. subtilis, and the genes ilvC plus ilvD from E.coli under the control of the promoter PLlacO1. The backbone of pJW12 is the plasmid pAM2991 with an NSI that can be integrated into the S. elongatus genomic DNA via homologous recombination (Fig. 1B). The wild-type S. elongatus was transformed with pAM2991 and pJW12, resulting in recombinant strains of JW10 and JW11, respectively. JW10 produced no observable isobutanol, while JW11 produced approximately 0.126 g/L of extracellular isobutanol within 20 days, which was similar to the unoptimized titers reported in several previous reports[16–18, 27]. Atsumi et al. achieved the highest titer (~ 0.5 g/L) for isobutanol so far without further modifying the engineered cyanobacteria and the medium. The slight variations for production titers ranged from 0.1–0.5 g/L, and this may have been due to the different cultivation conditions, such as different light intensities, inorganic carbon concentrations, aeration rates, and other factors.
The environmental stress impacts on isobutanol production of engineered S. elongatus were then examined. Nitrogen depletion, which has been reported to enhance the lipid accumulation of microalgae, was first investigated. However, a 70% reduction of NaNO3 in the BG11 medium apparently inhibited growth of the JW11 strain even though it was inoculated with cultures to maintain the initial OD730 of 1.5, thus the isobutanol production titer was not enhanced (data not shown). Then high salinity was applied by adding 2% sea salt into the BG11 medium. Although the final cell density was only ~ 60% of that without the addition of sea salt (Fig. 1C), the isobutanol titer was elevated by five-fold, reaching 0.637 g/L within 20 days (Fig. 1D). Considering the sea salt used was composed of 73.6% of NaCl and 26.4% of other elements, 1.47% NaCl was also added to the medium for JW11 cultivation to confirm that the sea salt-stimulated effect for isobutanol production was attributed to NaCl. A similar isobutanol titer, as when the 2% sea salt was used, was observed (Fig. 1D), indicating that osmotic stress was the primary contribution. In previously, modifying genotype of engineered cyanobacteria for enhancing carbon flux to isobutanol were commonly used strategies, except that Miao et al. recently reported that an adjustment of the media pH to neutral during cultivation can significantly improve the isobutanol titer to 0.435 g/L at day 40. Here we found another considerably simpler way could be used to enhance isobutanol production for engineered cyanobacteria compared with modifying the genotype of the host.
High salinity stimulated isobutanol production by altering metabolic profile to accumulate redox equivalents
To explore why osmotic stress stimulated isobutanol production, an untargeted metabolomics analysis for the strain JW11 was performed under a high-salinity condition and not. A total of 105 metabolites were annotated and quantified based on the LC-ESI-MS/MS spectra (Table S2). Metabolites with univariate statistical significance (fold change > 2.0 and p < 0.05) were selected for further analysis. A total of 21 and 32 differentially accumulated metabolites were identified between the control and the stressed cells in the ESI+ and ESI− modes, respectively. A total of 43 differentially accumulated metabolites were functionally localized in the central metabolic pathway of the cyanobacteria, as shown in Fig. 2A.
Among the 43 critical metabolites, R5P, F6P, and Ru5P were identified from the Calvin cycle, through which cyanobacteria are employed for carbon assimilation, and these were down-regulated by 12.5-fold, 2.4-fold, and 2.2-fold, respectively, under osmotic stress. Citrate and succinate from the TCA cycle required for aerobic respiration were down-regulated by 1.4-fold and 1.6-fold, respectively. A weakening of the Calvin cycle and TCA cycle explained cyanobacterial growth inhibition under the high salinity condition. On the other hand, Biosynthesis metabolisms were also attenuated due to osmotic stress. For example, four amino acids were identified and down-regulated by at least 1.6-fold under the high salinity condition. The abundances of six dipeptides showed dramatic reductions, indicating that the synthesis of proteins were also inhibited. Moreover, purine and pyrimidine metabolism, derived from R5P and glutamate, respectively, and responsible for nucleic acid synthesis, were strongly suppressed under osmotic stress. In contrast to eukaryotic microalgae, lipids were not observed to accumulate under osmotic stress in S. elongatus, whereas the abundance of lipids decomposed products, such as 3-hydroxybutyrate and isobutyrylglycine, increased by 15.5- and 3.65-fold, respectively. Reasonably, the abundance of acetylcarnitine, which is a lipid transporter for aerobic respiration, was also increased by 9.33-fold under the high-salinity condition, confirming the acceleration of lipid decomposition. Interestingly, the abundance of NADH increased by 5.1-fold under osmotic stress (Fig. 2). It is unlikely that the TCA cycle contributed to the extra NADH since TCA cycle was inhibited under osmotic stress. The enhanced lipids decomposition, which is also a pool for NADH in vivo, probably resulted in the increasing abundances of NADH. It is reasonable to speculate that cyanobacteria employed the lipids decomposition to provide a sufficient redox equivalent, such as NADH, for aerobic respiration due to inhibition of the TCA cycle. It is worth mentioning that one of the exogenous enzymes that was introduced for isobutanol biosynthesis, AdhA, was exactly a NADH-dependent enzyme. Therefore, it was speculated that the elevated NADH abundance was likely one of the reasons for the isobutanol production enhancement under osmotic stress. Additionally, some osmo-protectants, such as sucrose, raffinose and galactinol, were significantly up-regulated by 20–100 fold under salt stress and consistent with previous reports. The antioxidants, glutathione (GSH) and reduced GSH (GSSG) were up-regulated by 3-fold (Fig. 2B), consistent with the results analyzed in biochemically (Fig. 3C), leading to reduced abundance of reactive oxygen species (ROS) (Fig. 3B). In eukaryotic microalgae, salt-induced ROS is one of the reasons for lipids accumulation . However, the antioxidative ability seems to be strengthen in S. elongatus under the high salinity condition, thereby no ROS and lipids were observed to be accumulated in salt-stressed cells. In brief, the metabolisms required for energy and synthesis were substantially attenuated under the high salinity condition. However, S. elongatus adopted other strategies, such as acceleration in lipids decomposition and improvement in antioxidative ability, to alleviate the high salinity stress.
To verify the hypothesis that enhanced isobutanol production was due to increasing NADH level in vivo, absolute quantification assays were performed for selected energy metabolites via LC-MS using known concentrations of chemicals as standards. Consistent with the untargeted metabolomics profiles, 3PG, R5P, F6P, as well as GAP, involved in the Calvin cycle, and α-ketoglutarate, fumarate, malarate, isocitrate, as well as succinate, involved in the TCA cycle, were all down-regulated under the high-salinity condition. The metabolites connecting the Calvin cycle and the TCA cycle, such as PEP and acetyl-CoA, were also down-regulated, whereas the isobutanol precursor, pyruvate, did not drop under the high salinity condition. The two redox equivalents in vivo, NADH and NADPH, were up-regulated by three-fold and 1.3-fold, respectively (Fig. 4). Notably, among the five exogenous enzymes introduced for isobutanol biosynthesis, AdhA is a NADH-dependent enzyme, and IlvC is a NADPH-dependent enzyme. Therefore, it can be tentatively concluded that the increasing pools for redox equivalents contributed to the enhanced isobutanol production titer. As noted in the discussion above, an enhancement of lipid decomposition probably resulted in the regeneration of NADH, but it is unclear how the changed metabolic profiles impact the NADPH pools. One possible interpretation is that S. elongatus synthetized more glutathione to cope with redox stress, and the accelerated conversion between GSH and GSSG result in not only reduction of ROS but also regeneration of NADPH (Fig. 3A).
Effect of salinity stress on cell morphology, membrane permeability and photosynthetic pigments
To gain more insights into how salinity stress impact physiological functions of the S. elongatus, we performed analysis for cell morphology, membrane permeability and photosynthetic pigments under the osmotic stress and not. Scanning electron microscopy (SEM) results showed that the osmotic-stressed cells became obviously wrinkled and slimmer with a rod-shaped cell′s diameter of 0.61 ± 0.04 µm, compared to a stress-free cell′s diameter of 0.81 ± 0.07 µm (Fig. 5A and 5B).
Since cell morphology is partially influenced by membrane layers which encased the microalgal cells, we characterized the membrane permeability by quantifying relative conductivity of membranes as previously reported. Figure 6C showed that the relative conductivities under the salinity stress were three-fold higher than that of no salt added in the first 10 days′cultivation, indicating that the membrane permeability increased significantly due to the osmotic pressure-induced cell′damage. Transmission electron microscopy (TEM) results were another evidence showing that the cell′membrane was partially disturbed by osmotic stress (Fig. 5C and 5D). The increased membrane permeability was probably another reason that resulting in the enhanced production titer as it facilitated leakage of isobutanol, thereby reduced the toxicity to cyanobacterial cells.
Photosynthetic pigments are key indicators for photosynthesis of autotrophic microalga. Figure 6A and 6B showed that abundances of chlorophyll-a (Chl-a), the leading light-harvesting pigment, remained stable under the salinity stress, indicating that photo-phosphorylation was not affected by the salinity stress, whereas abundances of carotenoids increased roughly 1.4-fold during 20 days′cultivation. Considering that several components of carotenoids, such as β-carotene and astaxanthin, are well-characterized antioxidants, this result supported the fact that antioxidative ability was improved under the salinity stress, thereby ROS was not observed to accumulate in vivo.
A novel biotechnology for mixing synthetic wastewater with seawater to produce isobutanol
Nitrogen and phosphorus elements are considered to be the most critical nutrient factors that affect the growth and metabolism of aquatic cyanobacteria. Wastewater typically contains different levels of nitrogen and phosphorus elements, which may provide necessary nutrients for cyanobacterial growth. In addition, seawater can provide the high salinity condition required for massively producing isobutanol using engineered S. elongatus. Consequently, the recent proposed biotechnology concerning the mixing of wastewater with seawater to produce bioenergy by microalgae offers an attractive way to cultivate the engineered S. elongatus for sustainable isobutanol production.
First, an artificial wastewater was synthesized as previously reported, containing all of the necessary elements for cyanobacterial growth. However, the synthetic wastewater was composed of either 17 mg/L of NH4+-N or 17 mg/L of NO3−-N designated as basic-NH4+ and basic-NO3−, respectively, far below the 247 mg/L of NO3−-N in the BG11 medium. Therefore, NH4+-N or NO3−-N sources were added to the wastewater, reaching final nitrogen concentrations equal to that of the BG11 medium, designated as high-NH4+ and high-NO3−, respectively. The phosphorus content in the basic synthetic wastewater was similar to that of the BG11. An artificial seawater was also synthesized typically containing 3.5% salt salinity. To maintain an optimal salinity of 2% for the isobutanol enhancement, the seawater and wastewater were mixed at a ratio of 1:0.75. The results showed that the wastewater of the basic-NH4+ and basic-NO3− could support the growth of the engineered S. elongatus until day 8 and 10, respectively, and both of them could produce isobutanol with maximal titers of ~ 0.1 g/L at day 20. The addition of NH4+-N to the basic-NH4+ significantly inhibited growth from day 0 to day 12, but produced 0.15 g/L of isobutanol at day 20. The addition of NO3−-N to the basic-NO3− supported growth of the engineered S. elongatus continuously, producing 0.413 g/L of isobutanol, which were similar to that of BG11 plus 2% sea salt (Fig. 7A and 7B). Nitrogen and phosphorus utilization were also measured for all the cultivations. As expected, the total nitrogen was nearly exhausted for the basic-NH4+ and basic-NO3− at day 10. Due to the addition of excessive NH4+-N and NO3−-N, 20–60 mg/L of total nitrogen were consumed by the engineered S. elongatus, while phosphorus utilizations were enhanced at least two-fold (Fig. 7C and 7D).
These results indicated that the basic synthetic wastewater containing 17 mg/L of NH4+-N or NO3−-N could support the engineered S. elongatus to produce isobutanol. Generally, wastewater of NH4+-N, such as municipal and farming wastewater, typically consists of at least 20 mg/L of nitrogen, which are able to meet the requirement for the cultivation of engineered S. elongatus. Nevertheless, these results demonstrated that NO3−-N is the most crucial factor that can massively affect the growth and isobutanol production of engineered S. elongatus. Wastewater containing high-strength NO3−-N is being produced by a variety of industries, such as fertilizer, explosives, and metal finishing[36, 37]. For example, the nitrate concentrations in stainless manufacturing and the fertilizer industry are normally in the range of 0.7–1.0 g/L, which can sufficiently support engineered S. elongatus to grow and produce isobutanol. High concentration of NH4+-N apparently inhibited cyanobacterial growth, as reported previously for other microalgal species. The molecular basis for ammonia-nitrogen inhibition of algal photosynthesis remains unclear. The best evidence indicates NH4+ uncoupling of the electron transport in photosystem II by the breakdown of proton gradients necessary to drive photophosphorylation or inhibition via NH4+ competition with H2O during oxidation reactions, leading to O2 evolution, or both[39, 40].