Deciphering the mechanistic role of Bacillus paramycoides (PM51) and Bacillus tequilensis (PM52) in bio-sorption and phyto-assimilation of Cadmium via Linum usitatissimum L. Seedlings.

Three Cd2+ resistant bacterium's minimal inhibition concentrations were assessed and their percentages of Cd2+ accumulation were determined by measurements using an atomic absorption spectrophotometer (AAS). The results revealed that two isolates Bacillus paramycoides (PM51) and Bacillus tequilensis (PM52), identified by 16S rDNA gene sequencing, showed a higher percentage of Cd2+ accumulation i.e., 83.78% and 81.79%, respectively. Moreover, both novel strains can tolerate Cd2+ levels up to 2000 mg/L isolated from district Chakwal. Amplification of the czcD, nifH, and acdS genes was also performed. Batch bio-sorption studies revealed that at pH 7.0, 1 g/L of biomass, and an initial 150 mg/L Cd2+ concentration were the ideal bio-sorption conditions for Bacillus paramycoides (PM51) and Bacillus tequilensis (PM52). The experimental data were fit to Langmuir isotherm measurements and Freundlich isotherm model R2 values of 0.999 for each of these strains. Bio sorption processes showed pseudo-second-order kinetics. The intra-diffusion model showed Xi values for Bacillus paramycoides (PM51) and Bacillus tequilensis (PM52) of 2.26 and 2.23, respectively. Different surface ligands, was investigated through Fourier-transformation infrared spectroscopy (FTIR). The scanning electron microscope SEM images revealed that after Cd2+ adsorption, the cells of both strains became thick, adherent, and deformed. Additionally, both enhanced Linum usitatissimum plant seed germination under varied concentrations of Cd2+ (0 mg/L, 250 mg/L,350 mg/L, and 500 mg/L). Current findings suggest that the selected strains can be used as a sustainable part of bioremediation techniques.

The phototrophic bacteria Rhodomicrobium spp. are novel chassis for bioplastic production.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable alternative to petroleum-based plastics. PHB production at industrial scales remains infeasible, in part due to insufficient yields and high costs. Addressing these challenges requires identifying novel biological chassis for PHB production and modifying known biological chassis to enhance production using sustainable, renewable inputs. Here, we take the former approach and present the first description of PHB production by two prosthecate photosynthetic purple non-sulfur bacteria (PNSB), Rhodomicrobium vannielii and Rhodomicrobium udaipurense. We show that both species produce PHB across photoheterotrophic, photoautotrophic, photoferrotrophic, and photoelectrotrophic growth conditions. Both species show the greatest PHB titers during photoheterotrophic growth on butyrate with dinitrogen gas as a nitrogen source (up to 44.08 mg/L), while photoelectrotrophic growth demonstrated the lowest titers (up to 0.13 mg/L). These titers are both greater (photoheterotrophy) and less (photoelectrotrophy) than those observed previously in a related PNSB, Rhodopseudomonas palustris TIE-1. On the other hand, we observe the highest electron yields during photoautotrophic growth with hydrogen gas or ferrous iron electron donors, and these electron yields were generally greater than those observed previously in TIE-1. These data suggest that non model organisms like Rhodomicrobium should be explored for sustainable PHB production and highlights utility in exploring novel biological chassis.

Improving bioplastic production by Rhodopseudomonas palustris TIE-1 using synthetic biology and metabolic engineering.

With the increasing demand for sustainably produced renewable resources, it is important to look towards microorganisms capable of producing bioproducts such as biofuels and bioplastics. Though many systems for bioproduct production are well documented and tested in model organisms, it is essential to look beyond to non-model organisms to expand the field and take advantage of metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple, non-sulfur autotrophic, and anaerobic bacterium capable of producing bioproducts that are comparable to their petroleum-based counterparts. To induce bioplastic overproduction, genes that might have a potential role in the PHB biosynthesis such as the regulator, phaR, and phaZ known for its ability to degrade PHB granules were deleted using markerless deletion. Mutants in pathways that might compete with polyhydroxybutyrate (PHB) production such as glycogen and nitrogen fixation previously created to increase n -butanol production by TIE-1 were also tested. In addition, a phage integration system was developed to insert RuBisCO (RuBisCO form I and II genes) driven by a constitutive promoter P aphII into TIE- 1 genome. Our results show that deletion of the phaR gene of the PHB pathway increases PHB productivity when TIE-1 was grown photoheterotrophically with butyrate and ammonium chloride (NH 4 Cl). Mutants unable to make glycogen or fix dinitrogen gas show an increase in PHB productivity under photoautotrophic growth conditions with hydrogen. In addition, the engineered TIE-1 overexpressing RuBisCO form I and form II produces significantly more polyhydroxybutyrate than the wild type under photoheterotrophy with butyrate and photoautotrophy with hydrogen. Inserting RuBisCO genes into TIE-1 genome is a more effective strategy than deleting competitive pathways to increase PHB production in TIE-1. The phage integration system developed for TIE-1 thus creates numerous opportunities for synthetic biology in TIE-1.

Electroactive biofilms: how microbial electron transfer enables bioelectrochemical applications.

Microbial biofilms are ubiquitous. In marine and freshwater ecosystems, microbe-mineral interactions sustain biogeochemical cycles, while biofilms found on plants and animals can range from pathogens to commensals. Moreover, biofouling and biocorrosion represent significant challenges to industry. Bioprocessing is an opportunity to take advantage of biofilms and harness their utility as a chassis for biocommodity production. Electrochemical bioreactors have numerous potential applications, including wastewater treatment and commodity production. The literature examining these applications has demonstrated that the cell-surface interface is vital to facilitating these processes. Therefore, it is necessary to understand the state of knowledge regarding biofilms' role in bioprocessing. This mini-review discusses bacterial biofilm formation, cell-surface redox interactions, and the role of microbial electron transfer in bioprocesses. It also highlights some current goals and challenges with respect to microbe-mediated bioprocessing and future perspectives.

n-Butanol production by Rhodopseudomonas palustris TIE-1.

Anthropogenic carbon dioxide (CO2) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO2 to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO2. However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO2, here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO2, solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

Photoferrotrophy and phototrophic extracellular electron uptake is common in the marine anoxygenic phototroph Rhodovulum sulfidophilum.

Photoferrotrophy allows anoxygenic phototrophs to use reduced iron as an electron donor for primary productivity. Recent work shows that freshwater photoferrotrophs can use electrons from solid-phase conductive substances via phototrophic extracellular electron uptake (pEEU), and the two processes share the underlying electron uptake mechanism. However, the ability of marine phototrophs to perform photoferrotrophy and pEEU, and the contribution of these processes to primary productivity is largely unknown. To fill this knowledge gap, we isolated 15 new strains of the marine anoxygenic phototroph Rhodovulum sulfidophilum on electron donors such as acetate and thiosulfate. We observed that all of the R. sulfidophilum strains isolated can perform photoferrotrophy. We chose strain AB26 as a representative strain to study further, and find that it can also perform pEEU from poised electrodes. We show that during pEEU, AB26 transfers electrons to the photosynthetic electron transport chain. Furthermore, systems biology-guided mutant analysis shows that R. sulfidophilum AB26 uses a previously unknown diheme cytochrome c protein, which we call EeuP, for pEEU but not photoferrotrophy. Homologs of EeuP occur in a range of widely distributed marine microbes. Overall, these results suggest that photoferrotrophy and pEEU contribute to the biogeochemical cycling of iron and carbon in marine ecosystems.

Magnetite nanoparticle anchored graphene cathode enhances microbial electrosynthesis of polyhydroxybutyrate by Rhodopseudomonas palustris TIE-1.

Microbial electrosynthesis (MES) is an emerging technology that can convert carbon dioxide (CO2) into value-added organic carbon compounds using electrons supplied from a cathode. However, MES is affected by low product formation due to limited extracellular electron uptake by microbes. Herein, a novel cathode was developed from chemically synthesized magnetite nanoparticles and reduced graphene oxide nanocomposite (rGO-MNPs). This nanocomposite was electrochemically deposited on carbon felt (CF/rGO-MNPs), and the modified material was used as a cathode for MES production. The bioplastic, polyhydroxybutyrate (PHB) produced by Rhodopseudomonas palustris TIE-1 (TIE-1), was measured from reactors with modified and unmodified cathodes. Results demonstrate that the magnetite nanoparticle anchored graphene cathode (CF/rGO-MNPs) exhibited higher PHB production (91.31 ± 0.9 mg l-1). This is ∼4.2 times higher than unmodified carbon felt (CF), and 20 times higher than previously reported using graphite. This modified cathode enhanced electron uptake to -11.7 ± 0.1 µA cm-2, ∼5 times higher than CF cathode (-2.3 ± 0.08 µA cm-2). The faradaic efficiency of the modified cathode was ∼2 times higher than the unmodified cathode. Electrochemical analysis and scanning electron microscopy suggest that rGO-MNPs facilitated electron uptake and improved PHB production by TIE-1. Overall, the nanocomposite (rGO-MNPs) cathode modification enhances MES efficiency.

Photoferrotrophs Produce a PioAB Electron Conduit for Extracellular Electron Uptake.

Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide using light energy. They can also use poised electrodes as their electron donor via phototrophic extracellular electron uptake (phototrophic EEU). The electron uptake mechanisms underlying these processes are not well understood. Using Rhodopseudomonas palustris TIE-1 as a model, we show that a single periplasmic decaheme cytochrome c, PioA, and an outer membrane porin, PioB, form a complex allowing extracellular electron uptake across the outer membrane from both soluble iron and poised electrodes. We observe that PioA undergoes postsecretory proteolysis of its N terminus to produce a shorter heme-attached PioA (holo-PioAC, where PioAC represents the C terminus of PioA), which can exist both freely in the periplasm and in a complex with PioB. The extended N-terminal peptide controls heme attachment, and its processing is required to produce wild-type levels of holo-PioAC and holo-PioACB complex. It is also conserved in PioA homologs from other phototrophs. The presence of PioAB in these organisms correlate with their ability to perform photoferrotrophy and phototrophic EEU.IMPORTANCE Some anoxygenic phototrophs use soluble iron, insoluble iron minerals (such as rust), or their proxies (poised electrodes) as electron donors for photosynthesis. However, the underlying electron uptake mechanisms are not well established. Here, we show that these phototrophs use a protein complex made of an outer membrane porin and a periplasmic decaheme cytochrome (electron transfer protein) to harvest electrons from both soluble iron and poised electrodes. This complex has two unique characteristics: (i) it lacks an extracellular cytochrome c, and (ii) the periplasmic decaheme cytochrome c undergoes proteolytic cleavage to produce a functional electron transfer protein. These characteristics are conserved in phototrophs harboring homologous proteins.

Phototrophic extracellular electron uptake is linked to carbon dioxide fixation in the bacterium Rhodopseudomonas palustris.

Extracellular electron uptake (EEU) is the ability of microbes to take up electrons from solid-phase conductive substances such as metal oxides. EEU is performed by prevalent phototrophic bacterial genera, but the electron transfer pathways and the physiological electron sinks are poorly understood. Here we show that electrons enter the photosynthetic electron transport chain during EEU in the phototrophic bacterium Rhodopseudomonas palustris TIE-1. Cathodic electron flow is also correlated with a highly reducing intracellular redox environment. We show that reducing equivalents are used for carbon dioxide (CO2) fixation, which is the primary electron sink. Deletion of the genes encoding ruBisCO (the CO2-fixing enzyme of the Calvin-Benson-Bassham cycle) leads to a 90% reduction in EEU. This work shows that phototrophs can directly use solid-phase conductive substances for electron transfer, energy transduction, and CO2 fixation.

Towards sustainable bioplastic production using the photoautotrophic bacterium Rhodopseudomonas palustris TIE-1.

Bacterial synthesis of polyhydroxybutyrates (PHBs) is a potential approach for producing biodegradable plastics. This study assessed the ability of Rhodopseudomonas palustris TIE-1 to produce PHBs under various conditions. We focused on photoautotrophy using a poised electrode (photoelectroautotrophy) or ferrous iron (photoferroautotrophy) as electron donors. Growth conditions were tested with either ammonium chloride or dinitrogen gas as the nitrogen source. Although TIE-1's capacity to produce PHBs varied fairly under different conditions, photoelectroautotrophy and photoferroautotrophy showed the highest PHB electron yield and the highest specific PHB productivity, respectively. Gene expression analysis showed that there was no differential expression in PHB biosynthesis genes. This suggests that the variations in PHB accumulation might be post-transcriptionally regulated. This is the first study to systematically quantify the amount of PHB produced by a microbe via photoelectroautotrophy and photoferroautotrophy. This work could lead to sustainable bioproduction using abundant resources such as light, electricity, iron, and carbon dioxide.

An insoluble iron complex coated cathode enhances direct electron uptake by Rhodopseudomonas palustris TIE-1.

Microbial electrosynthesis (MES) is a promising bioelectrochemical approach to produce biochemicals. A previous study showed that Rhodopseudomonas palustris TIE-1 can directly use poised electrodes as electron donors for photoautotrophic growth at cathodic potentials that avoid electrolytic H2 production (photoelectroautotrophy). To make TIE-1 an effective biocatalyst for MES, we need to improve its electron uptake ability and growth under photoelectroautotrophic conditions. Because TIE-1 interacts with various forms of iron while using it as a source of electrons for photoautotrophy (photoferroautotrophy), we tested the ability of iron-based redox mediators to enhance direct electron uptake. Our data show that soluble iron cannot act as a redox mediator for electron uptake by TIE-1 from a cathode poised at +100mV vs. Standard Hydrogen electrode. We then tested whether an immobilized iron-based redox mediator Prussian blue (PB) can enhance electron uptake by TIE-1. Chronoamperometry indicates that cathodic current uptake by TIE-1 increased from 1.47±0.04 to 5.6±0.09µA/cm2 (3.8 times). Overall, our data show that immobilized PB can enhance direct electron uptake by TIE-1.

Simultaneous degradation of bad wine and electricity generation with the aid of the coexisting biocatalysts Acetobacter aceti and Gluconobacter roseus.

This study describes the cooperative effect of the two biocatalysts Acetobacter aceti and Gluconobacter roseus for biodegradation as well as current generation. The electro activity of the biofilms of these two microorganisms was investigated by the bioelectrocatalytic oxidation of ethanol and glucose using cyclic voltammetry. Two chamber microbial fuel cells (MFCs) were constructed using single culture of A. aceti (A-MFC), and G. roseus (G-MFC) and also using mixed culture (AG-MFC). Each MFC was fed with four different substrates viz., glucose, ethanol, acetate and bad wine. AG-MFC produced higher power density with glucose (1.05 W/m(3)), ethanol (1.97 W/m(3)), acetate (1.39 W/m(3)) and bad wine (3.82 W/m(3)). COD removal (94%) was maximum for acetate fed MFCs. Higher coulombic efficiency was obtained with bad wine (45%) as the fuel. This work provides the scope of using these biofuel cells in wineries for performing the dual duty of bad wine degradation along with current generation.