Protocol to analyze the inhibitory effect of free organic carbon generated by chemoautotrophic bacteria on their CO2 fixation.

Here, we present a protocol to analyze the inhibition of self-generated extracellular free organic carbon (EFOC) on CO2 fixation by chemoautotrophic bacteria. We detail the construction and operation of membrane reactor, followed by a simulation experiment to verify the inhibition of EFOC on CO2 fixation. We further describe the analysis of main inhibitory components in EFOC and measurement of abundance and transcription level of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) gene to clarify the mechanism of the main inhibition components on CO2 fixation. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Cadmium stimulated cooperation between bacterial endophytes and plant intrinsic detoxification mechanism in Lonicera japonica thunb.

Due to the intimate association between plant physiology and metabolism, the internal colonizing microbe (endophytes) community must be adjusted to support plant productivity in response to cell damage in plants under stress. However, how endophytes coordinate their activities with plant intrinsic mechanisms such as antioxidative systems and detoxification pathways during Cd accumulation remains unknown. In this hydroponic pot study, we investigated how exposure of Lonicera japonica. thunb. to different levels of Cd (0.5, 2.5, 5, 10, and 20 mg kg-1) affected plant growth, metabolic pathways, and endophyte community structure and function. Although Cd accumulation increased at 5 mg kg-1 Cd, the biomass and height of L. japonica increased in association with elevated endophyte-involved plant detoxification activities. Endophytes, such as Sphingomonas, Klenkia, and Modestobacter, expressed major antioxidative regulators (superoxide dismutase and ascorbate acid) to detoxify Cd in L. japonica. Furthermore, L. japonica and its endophytes synergistically regulated the toxic effects of Cd accumulation via multiple plant metabolic defensive pathways to increase resistance to metal-induced stress.

Continuous-flow membrane bioreactor enhances enrichment and culture of autotrophic nitrifying bacteria by removing extracellular free organic carbon.

An activated sludge system can be inoculated with enriched nitrifying bacteria to enhance NH4+-N removal, or enriched nitrifying bacteria can be added directly to a river to remove NH4+-N. However, the enrichment culture is still generally inefficient and the technical bottleneck has not been clarified. Previous studies have shown that extracellular free organic carbon (EFOC) inhibits the growth of some autotrophic bacteria, and separating EFOC during culture with a membrane bioreactor (MBR) promotes the continuous growth of autotrophic bacteria and CO2 fixation. However, whether a membrane bioreactor can also be used to enrich and culture autotrophic nitrifying bacteria by separating EFOC has not been verified. In this study, an MBR was constructed to separate EFOC during the culture of nitrifying bacteria in activated sludge to confirm that the MBR better enriches and cultures nitrifying bacteria than a sequencing batch reactor (SBR). Our results showed that after culture for 34 days, the rate of NH4+-N removal and the nitrification rate by nitrifying bacteria in the MBR were 2.20-fold and 1.42-fold higher than in the SBR, respectively. The abundance of Nitrospira in the MBR was also 7.23-fold greater than in the SBR at the end of the experimental period. After 34 days, the average concentration of EFOC and the average EFOC/bacterial organic carbon ratio in the MBR were only 53% and 37% of those in the SBR, respectively. A correlation analysis suggested that the timely removal by the MBR of the EFOC generated during the culture process may be an important factor in promoting the growth of autotrophic nitrifying bacteria. The possible mechanism by which the MBR separates EFOC to the growth of promote autotrophic nitrifying bacteria is discussed from the perspective of the inhibitory effect of EFOC on cbb gene transcription. Our experimental results suggest a new approach to enhancing the enrichment of autotrophic nitrifying bacteria and extending the application of MBRs.

Main components of free organic carbon generated by obligate chemoautotrophic bacteria that inhibit their CO2 fixation.

Chemoautotrophic bacteria play an important role in combating the rise in global CO2. However, recently it was found that extracellular free organic carbon (EFOC) generated by chemoautotrophic bacteria inhibits their CO2 fixation. Although continuous-flow membrane bioreactor can remove EFOC and enrich bacteria, it may also remove beneficial bio-factors for bacterial growth. Finding out the main inhibitory factors and inhibitory mechanisms in EFOC can provide theoretical guidance for the development of targeted inhibitory component removal technology. The results show a significant negative correlation between the increasing proportion of small-molecule EFOC and the decreasing trend of CO2 fixation efficiency, and simulation experiments confirm that the small molecule organics such as amino acids and organic acids are the main components of EFOC that inhibit CO2 fixation by inhibiting ribulose bisphosphate carboxylase/oxygenase (RuBisCO) gene (cbb) transcription efficiency. Therefore, amino acids and organic acids are suggested to be recovered to promote efficient CO2 fixation of autotrophic bacteria.

Mixed electron donors synergistically enhance CO2 fixation of non-photosynthetic microorganism communities through optimizing community structure to promote cbb gene transcription.

Studies have shown that mixed electron donors (MEDs) can enhance the CO2-fixing efficiency of non-photosynthetic microbial communities (NPMCs), even up to the level of fixation observed when H2 is used as an electron donor. However, this promotion effect is not stable because its mechanism remains unclear. To elucidate the mechanisms involved, allowing further regulation and optimization of the MED system for improving the CO2-fixing efficiency of NPMCs consistently, cbb gene transcription level and efficiency, extracellular free organic carbon (EFOC) content as well as microbial structure of NPMCs under MED and other electron donor systems were investigated. MEDs synergistically promoted CO2 fixation efficiency of NPMCs, even producing levels seen when H2 was used as the electron donor. Subsequent experiments revealed that the cbb gene abundance and transcription level in the MED system were high compared with those in other single-electron donor systems; the concentration of EFOC per unit cell was relatively lower than that in any other electron donor system; and the system developed a large number of dominant heterotrophic bacteria such as Enterobacteriaceae and Vibrionaceae. Data analysis revealed a high negative correlation between EFOC concentration per unit cell and cbb gene abundance as well as gene transcription level. These results implied that MEDs can promote a complex microbial community structure enriched with high-efficiency heterotrophic bacteria, which can effectively reduce excessive EFOC generated by NPMCs in the CO2 fixation process, promoting overall cbb gene abundance and transcription level within the NPMC and thus enhancing CO2 fixation.

A continuous flow membrane bio-reactor releases the feedback inhibition of self-generated free organic carbon on cbb gene transcription of a typical chemoautotrophic bacterium to improve its CO2 fixation efficiency.

Since the free organic carbon (FOC) generated by chemoautotrophic bacterium self has a feedback inhibition effect on its growth and carbon fixation, a continuous flow membrane bio-reactor was designed to remove extracellular FOC (EFOC) and release its inhibition effect. The promotion effect of membrane reactor on growth and carbon fixation of typical chemoautotrophic bacterium and its mechanism were studied. The accumulated apparent carbon fixation yield in membrane reactor was 3.24 times that in the control reactor. The EFOC per unit bacteria and cbb gene transcription level in membrane reactor were about 0.41 times and 11.18 times that in control reactor in late stage, respectively. Membrane reactor separated out EFOC, especially the small molecules, which facilitated the release of intracellular FOC, thereby releasing the inhibition of FOC on cbb gene transcription, thus promoting growth and carbon fixation of the typical chemoautotrophic bacterium. This study lays a foundation for enhancing carbon fixation by chemoautotrophic bacteria and expands the application field of membrane reactor.

Utilization of the saccharification residue of rice straw in the preparation of biochar is a novel strategy for reducing CO2 emissions.

Once rice straw has been bioconverted into biofuels, it is difficult to further biodegrade or decompose the saccharification residue (mainly lignin). Taking into account the pyrolysis characteristics of lignin, in this study the saccharification residue was used as a raw material for the preparation of biochar (biochar-SR), a potential soil amendment. Biochar was prepared directly from rice straw (biochar-O) with a yield of 32.45 g/100 g rice straw, whereas 30.14 g biochar-SR and 30.46 g monosaccharides (including 20.46 g glucose, 9.11 g xylose, and 0.89 g arabinose) were obtained from 100 g of rice straw. When added to liquid soil extracts as a soil amendment, almost nothing was released from biochar-SR, whereas numerous dissolved solids (about 70 mg/L) were released from biochar-O. Adding a mixture of biochar-SR and autotrophic bacteria improved soil total organic carbon 1.8-fold and increased the transcription levels of cbbL and cbbM, which were 4.76 × 103 and 3.76 × 105 times those of the initial blank, respectively. By analyzing the soil microbial community, it was clear that the above mixture favored the growth of CO2-fixing bacteria such as Ochrobactrum. Compared with burning rice straw or preparing biochar-O, the preparation of biochar-SR reduced CO2 emissions by 67.53% or 37.13%, respectively. These results demonstrate that biochar-SR has potential applications in reducing the cost of sustainable energy and addressing environmental issues.

Role of electron donor in CO2 fixation of chemoautotrophic bacteria and its preconditions: Verification in Alcaligenes hydrogenophilus.

Alcaligenes hydrogenophilus was used to verify the role of the electron donor and acceptor in apparent CO2 fixation of chemoautotrophic bacteria. The response mechanisms underlying the apparent CO2 fixation characteristics with different concentrations of electron donor and acceptor were elucidated by analyzing the transcription characteristics of the cbbL gene, cytoskeleton synthesis efficiency and extracellular free organic carbon concentration. The results showed that the apparent CO2 fixation efficiency of A. hydrogenophilus was significantly influenced by the electron donor (H2), and the degree of electron donor oxidization was responsible for the variation in apparent CO2 fixation efficiency. Furthermore, transcription efficiency of the cbbL gene at low electron donor concentration was lower than that at high electron donor concentration, but excessive electron donor concentrations did not further increase cbbL gene transcription efficiency significantly. High oxygen concentration was not advantageous to cbbL gene transcription efficiency in A. hydrogenophilus, but could improve cell growth rate (protein synthesis rate) and apparent CO2 fixation efficiency, implying that cytoskeleton synthesis efficiency is another important factor determining apparent CO2 fixation efficiency and its contribution maybe greater than that of cbbL transcription. The results also indicated that high apparent CO2 fixation efficiency required the matching of electron donor and acceptor.