Limiting Carbon Source Research Articles

Hydrogen Generated by Electrochemical Water Splitting as Electron Donor for Nitrate Removal from Micro-Polluted Reservoir Water

Extremely limited organic carbon sources and aerobic environment in micro-polluted reservoir water make conventional denitrification exceptionally challenging. As a result, total nitrogen (TN) concentration in most reservoir waters exceeds standard value year-round. In this study, for the first time, we constructed a mini water-lifting and aeration system (mini-WLAS) to remove nitrate in actual reservoir water. In the mini-WLAS, H2 was produced through electrolysis of reservoir water without adding any electrolyte, and the ascending water flow carried the generated H2 from lower layer to upper bacteria layer. The maximum denitrification rate reached 0.29mg (L·d)-1 under dissolved oxygen (DO) concentration of 6-8mgL-1, 6.04 times higher than that of the control group. There is almost no accumulation of NH4+-N, NO2--N, and N2O, and the concentration of CODMn decreased by 55.2%. More importantly, the pH stayed near-neutral steadily throughout the whole process. Microbial community analysis showed that the abundances of hydrogenotrophic denitrifying bacteria (HDB) were 2 orders higher than those in the control system. Some HDB could work under aerobic conditions, providing an explanation for the excellent denitrification performance under high DO. This study provides a novel perspective for TN removal from reservoir water.

Increasing biomass concentration facilitates simultaneous nitrogen removal and sludge reduction under low C/N conditions

To overcome the issues of limited carbon source and high sludge production in partial denitrification/anammox (PD/A) process, the effects of mixed liquor suspended solids (MLSS) and carbon/nitrogen ratio (C/N) on PD/A were investigated through parallel experiments. Nitrogen removal efficiencies decreased significantly when C/N was reduced (1.5 → 0.75). When MLSS was doubled, the nitrogen removal efficiencies in the two parallel reactors increased from 75.3 %, 72.9 % to 86.9 %, 89.7 %, respectively, and sludge yields decreased obviously. Combining with in-situ test, it was speculated when MLSS increased, fermentation was enhanced, providing substrate for partial denitrification. Thauera, involved in partial denitrification, decreased obviously with reduced C/N, but increased from 9.93 % to 38.16 % when MLSS doubled, which could promote the PD/A process. Terrimonas and Ignavibacterium (fermentative bacteria) increased from 1.26 %, 5.22 % to 6.62 %, 6.30 %, respectively. These results proved that increasing MLSS under low C/N ratios promoted fermentation in PD/A system, facilitating efficient nitrogen removal and sludge reduction.

Integrated process of Tetrasphaera-dominated in-situ waste activated sludge fermentation, biological phosphorus removal and endogenous denitrification in single reactor for treatment of wastewater with limited carbon sources

An integrated process of sludge in-situ fermentation, biological phosphorus removal and endogenous denitrification (ISFPR-ED) was developed to treat low ratio of chemical oxygen demand to nitrogen (COD/N) wastewater and waste activated sludge (WAS) in a single reactor. Nutrient removal and WAS reduction were achieved due to Tetrasphaera-dominated sludge fermentation provided organic carbon in extending the anaerobic duration. The WAS reduction efficiency, effluent orthophosphate (PO43−-P) and total inorganic nitrogen reached 28.1 %, less than 0.4 and 7.2 mg/L, respectively. While organic carbon was reduced by 67 %. Tetrasphaera, conventional polyphosphate accumulating organisms (PAOs) stored glycogen, amino acids, and PHA for nutrient removal. Excess energy from fermentation enhanced anaerobic PO43−-P uptake by Tetrasphaera. Tetrasphaera was the dominant PO43−-P removal and fermentation bacteria, working synergistically with conventional PAOs and fermenting microorganisms. This integrated process improves nutrient removal efficiency and reduces operating costs for carbon addition and WAS disposal in wastewater treatment.

Living in mangroves: a syntrophic scenario unveiling a resourceful microbiome

BackgroundMangroves are complex and dynamic coastal ecosystems under frequent fluctuations in physicochemical conditions related to the tidal regime. The frequent variation in organic matter concentration, nutrients, and oxygen availability, among other factors, drives the microbial community composition, favoring syntrophic populations harboring a rich and diverse, stress-driven metabolism. Mangroves are known for their carbon sequestration capability, and their complex and integrated metabolic activity is essential to global biogeochemical cycling. Here, we present a metabolic reconstruction based on the genomic functional capability and flux profile between sympatric MAGs co-assembled from a tropical restored mangrove.ResultsEleven MAGs were assigned to six Bacteria phyla, all distantly related to the available reference genomes. The metabolic reconstruction showed several potential coupling points and shortcuts between complementary routes and predicted syntrophic interactions. Two metabolic scenarios were drawn: a heterotrophic scenario with plenty of carbon sources and an autotrophic scenario with limited carbon sources or under inhibitory conditions. The sulfur cycle was dominant over methane and the major pathways identified were acetate oxidation coupled to sulfate reduction, heterotrophic acetogenesis coupled to carbohydrate catabolism, ethanol production and carbon fixation. Interestingly, several gene sets and metabolic routes similar to those described for wastewater and organic effluent treatment processes were identified.ConclusionThe mangrove microbial community metabolic reconstruction reflected the flexibility required to survive in fluctuating environments as the microhabitats created by the tidal regime in mangrove sediments. The metabolic components related to wastewater and organic effluent treatment processes identified strongly suggest that mangrove microbial communities could represent a resourceful microbial model for biotechnological applications that occur naturally in the environment.

Effect of microbial growth and electron competition on nitrous oxide source and sink function of hyporheic zones

The hyporheic zones (HZs) are key sites of the production of nitrous oxide (N2O), a potent ozone-depleting greenhouse gas. Denitrification is the primary process of N2O production in HZs, including four reduction steps (NO3−→NO2−→NO→N2O→N2). Electron competition occurs between the four reduction steps and can significantly impact the production of N2O. However, denitrification was typically considered as simplified two step reactions for investigating the release of N2O, neglecting the electron competition among the four-step reactions. Moreover, the N2O production/consumption patterns are regulated by both hydraulic and biogeochemical conditions in HZs. Dynamic microbial growth cannot only mediate the biogeochemical reactions, but also change hydraulic properties spatiotemporally by bioclogging. But microbial growth is rarely considered for investigating N2O dynamics of HZs. To assess these effects on hyporheic N2O dynamics and source-sink function, we establish a novel numerical model of N2O dynamics of HZs, coupling porous flow, reactive transport, electron competition, microbial growth and bioclogging. The results show that the weak electron competitiveness of N2O reductase results in a less allocation of electrons to the N2O reduction process, particularly in situations with limited carbon sources, thus increasing the release of N2O into the rives. Microbial growth significantly influences N2O release from HZs into rivers, increasing by more than two orders of magnitude on average compared to the model neglecting microbial dynamics. In contrast to the classical knowledges that HZs in coarse sediments tending to short residence time cannot act as sources of N2O, dynamic microbial growth obviously increases the potential for N2O release from HZs in coarse sediments to the rivers. The global Monte Carlo regional sensitivity analyses indicate that microbial biomass is the most critical factor determined the hyporheic source-sink function for N2O, followed by carbon oxidation rate and residence time. These are significantly different from previous knowledge that the residence time and oxygen/nitrogen uptake rate are the most sensitive parameters, which may lead to misunderstanding of the key controlling factors of N2O release from HZs. In addition, we propose a new Damköhler number (DaO2∗) of dissolved oxygen by multiplying the classical DaO2 with a dimensionless microbial modification factor for identifying N2O source-sink function of HZs, with DaO2∗ < 1 for N2O sink, while DaO2∗ > 1 for N2O source.

A commercial humic acid inhibits benzo(a)pyrene biodegradation by Paracoccus aminovorans HPD-2

Benzo(a)pyrene (BaP) is posing serious threats to soil ecosystems and its bioremediation usually limited by environmental factors and microbial activity. Humic acid (HA), a ubiquitous heterogeneous organic matter, which could affect the fate of environmental pollutants. However, the impact of HA on bioremediation of organic contamination remains controversial. In the present study, the biodegradation of BaP by Paracoccus aminovorans HPD-2 with and without HA was explored. Approximately 87.4 % of BaP was biodegraded in the HPD-2 treatment after 5 days of incubation, whereas the addition of HA dramatically reduced BaP biodegradation to 56.0 %. The limited BaP biodegradation in the HA + HPD-2 treatment was probably due to the decrease of BaP bioavailability which induced by the adsorption of HA with unspecific interactions. The excitation-emission matrix (EEM) of fluorescence characteristics showed that strain HPD-2 was responsible for the presence of protein-like substances and the microbial original humic substances in the HPD-2 treatment. Addition of HA would result in the increase of soluble microbial humic-like material, which should ascribe to the biodegradation of BaP and probably utilization of HA. Furthermore, both the growth and survival of strain HPD-2 were inhibited in the HA + HPD-2 treatment, because of the limited available carbon source (i.e. BaP) at the presence of HA. The expression of gene1789 and gene2589 dramatically decreased in the HA + HPD-2 treatment, and this should be responsible for the decrease of BaP biodegradation as well. This study reveals the mechanism that HA affect the BaP biodegradation, and the decrease of biodegradation should ascribe to the interaction of HA and bacterial strain. Thus, the bioremediation strategies of PAHs need to consider the effects of organic matter in environment.

Enhancing nitrogen removal in constructed wetlands: The role of influent substrate concentrations in integrated vertical-flow systems

Recent advancements in constructed wetlands (CWs) have highlighted the imperative of enhancing nitrogen (N) removal efficiency. However, the variability in influent substrate concentrations presents a challenge in optimizing N removal strategies due to its impact on removal efficiency and mechanisms. Here we show the interplay between influent substrate concentration and N removal processes within integrated vertical-flow constructed wetlands (IVFCWs), using wastewaters enriched with NO3−-N and NH4+-N at varying carbon to nitrogen (C/N) ratios (1, 3, and 6). In the NO3−-N enriched systems, a positive correlation was observed between the C/N ratio and total nitrogen (TN) removal efficiency, which markedly increased from 13.46 ± 2.23% to 87.00 ± 2.37% as the C/N ratio escalated from 1 to 6. Conversely, in NH4+-N enriched systems, TN removal efficiencies in the A-6 setup (33.69 ± 4.83%) were marginally 1.25 to 1.29 times higher than those in A-3 and A-1 systems, attributed to constraints in dissolved oxygen (DO) levels and alkalinity. Microbial community analysis and metabolic pathway assessment revealed that anaerobic denitrification, microbial N assimilation, and dissimilatory nitrate reduction to ammonium (DNRA) predominated in NO3−-N systems with higher C/N ratios (C/N ≥ 3). In contrast, aerobic denitrification and microbial N assimilation were the primary pathways in NH4+-N systems and low C/N NO3−-N systems. A mass balance approach indicated denitrification and microbial N assimilation contributed 4.12–47.12% and 8.51–38.96% in NO3−-N systems, respectively, and 0.55–17.35% and 7.83–33.55% in NH4+-N systems to TN removal. To enhance N removal, strategies for NO3−-N dominated systems should address carbon source limitations and electron competition between denitrification and DNRA processes, while NH4+-N dominated systems require optimization of carbon utilization pathways, and ensuring adequate DO and alkalinity supply.

ASIA: An automated stress-inducible adaptor for enhanced stress protein expression in engineered Escherichia coli.

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a potent technique for protein expression in broad cells, but the energy requirements associated with this method impede the growth, leading to cell lysis when dealing with toxic and stress proteins. A Lemo21(DE3) strain denoted as L21 offers a solution by fine-tuning T7RNAP levels under rhamnose to induce T7 lysozyme (LysY) and enhance the protein production, but it requires optimization of inducer concentration, cultural temperature, and condition, even the types of carbon sources. Herein, we construct an automated stress-inducible adaptor (ASIA) employing different stress-inducible promoters from Escherichia coli. The ASIA system is designed to automatically regulate LysY expression in response to stress signals, thereby suppressing T7RNAP and amplifying the overexpression of stress protein cutinase ICCM. This approach fine-tunes T7RNAP levels and outperforms L21 in various temperatures and carbon source conditions. The ASIAhtp strain maintains ICCM yield at 91.6 mg/g-DCW even in the limiting carbon source at 1 g/L, which is 12-fold higher in protein productivity compared to using L21. ASIA as a versatile and robust tool for enhancing overexpression of stress proteins in E. coli is expected to address more difficult proteins in the future.

Revealing zooplankton diversity in the midnight zone

Zooplankton diversity in the deep “midnight zone” (&amp;gt;1000 m), where sunlight does not reach, remains largely unknown. Uncovering such diversity has been challenging because of the major difficulties in sampling deep pelagic fauna and identifying many (unknown) species that belong to these complex swimmer assemblages. In this study, we evaluated zooplankton diversity using two taxonomic marker genes: mitochondrial cytochrome oxidase subunit 1 (COI) and nuclear 18S ribosomal RNA (18S). We collected samples from plankton net tows, ranging from the surface to a depth of 5000 m above the Atacama Trench in the Southeast Pacific. Our study aimed to assess the zooplankton diversity among layers from the upper 1000 m to the ultra-deep abyssopelagic zone to test the hypothesis of decreasing diversity with depth resulting from limited carbon sources. The results showed unique, highly vertically structured communities within the five depth strata sampled, with maximal species richness observed in the upper bathypelagic layer (1000–2000 m). The high species richness of zooplankton (&amp;gt;750 OTUS) at these depths was higher than that found in the upper 1000 m. The vertical diversity trend exhibited a pattern similar to the well-known vertical pattern described for the benthic system. However, a large part of this diversity was either unknown (&amp;gt;50%) or could not be assigned to any known species in current genetic diversity databases. DNA analysis showed that the Calanoid copepods, mostly represented by Subeucalanus monachus, the Euphausiacea, Euphausia mucronata, and the halocypridade, Paraconchoecia dasyophthalma, dominated the community. Water column temperature, dissolved oxygen, particulate carbon, and nitrogen appeared to be related to the observed vertical diversity pattern. Our findings revealed rich and little-known zooplankton diversity in the deep sea, emphasizing the importance of further exploration of this ecosystem to conserve and protect its unique biota.

Multifaceted benefits of magnesium hydroxide dosing in sewer systems: Impacts on downstream wastewater treatment processes

Magnesium hydroxide [Mg(OH)2] is a non-hazardous chemical widely applied in sewer systems for managing odour and corrosion. Despite its proven effectiveness in mitigating these issues, the impacts of dosing Mg(OH)2 in sewers on downstream wastewater treatment plants have not been comprehensively investigated. Through a one-year operation of laboratory-scale urban wastewater systems, including sewer reactors, sequencing batch reactors, and anaerobic sludge digesters, the findings indicated that Mg(OH)2 dosing in sewer systems had multifaceted benefits on downstream treatment processes. Compared to the control, the Mg(OH)2-dosed experimental system displayed elevated sewage pH (8.8±0.1vs 7.1±0.1), reduced sulfide concentration by 35.1%±4.9% (6.7±0.9mgSL−1), and lower methane concentration by 58.0%±4.9% (19.1±3.6mgCODL−1). Additionally, it increased alkalinity by 16.3%±2.2% (51.9±5.4mgCaCO3L−1), and volatile fatty acids concentration by 207.4%±22.2% (56.6±9.0mgCODL−1) in sewer effluent. While these changes offered limited advantages for downstream nitrogen removal in systems with sufficient alkalinity and carbon sources, significant improvements in ammonium oxidation rate and NOx reduction rate were observed in cases with limited alkalinity and carbon sources availability. Moreover, Mg(OH)2 dosing in upstream did not have any detrimental effects on anaerobic sludge digesters. Magnesium-phosphate precipitation led to a 31.7%±4.1% reduction in phosphate concertation in anaerobic digester sludge supernatant (56.1±10.4mgPL−1). The retention of magnesium in sludge increased settleability by 13.9%±1.6% and improved digested sludge dewaterability by 10.7%±5.3%. Consequently, the use of Mg(OH)2 dosing in sewers could potentially reduce downstream chemical demand and costs for carbon sources (e.g., acetate), pH adjustment and sludge dewatering.

Municipal wastewater treatment with limited carbon sources: The significance and advantages of transformation from partial denitrification to partial nitrification coupling with anammox

Anaerobic ammonium oxidation (anammox) combined with partial nitrification (PN) is expected to achieve energy self-sufficiency in wastewater treatment, while its advantages have not been fully revealed. An anaerobic/aerobic/anoxic (AOA) system was established to treat low chemical oxygen demand (COD)-total inorganic nitrogen (TIN) (C/N) ratio (3.08) municipal wastewater. Anammox bacteria were enriched from 7 × 106 to 3 × 108 copies/gSS during partial denitrification coupling with anammox (PDA) phase. With the PN achievement, the anammox hotspot zone transformed from the anoxic zone through PDA (37%) to the aerobic zone by partial nitrification coupled with anammox (PNA) (12%). As the nitrite accumulation rate increased to 97%, the TIN removal efficiency and rate increased to 95% and 0.12 kgN/(m3·d). Although anammox performance weakened after this transformation, the carbon sources saved by PN drove more nitrogen removal. PNA was beneficial for advanced and efficient nitrogen removal in carbon-limited wastewater treatment and was less dependent on higher anammox contribution.

Fungal Ligninolytic Enzymes and Their Application in Biomass Lignin Pretreatment.

Lignocellulosic biomass is a significant source of sustainable fuel and high-value chemical production. However, due to the complex cross-linked three-dimensional network structure, lignin is highly rigid to degradation. In natural environments, the degradation is performed by wood-rotting fungi. The process is slow, and thus, the use of lignin degradation by fungi has not been regarded as a feasible technology in the industrial lignocellulose treatment. Fungi produce a wide variety of ligninolytic enzymes that can be directly introduced in industrial processing of lignocellulose. Within this study, screening of ligninolytic enzyme production using decolorization of ABTS and Azure B dyes was performed for 10 fungal strains with potentially high enzyme production abilities. In addition to standard screening methods, media containing lignin and hay biomass as carbon sources were used to determine the change in enzyme production depending on the substrate. All selected fungi demonstrated the ability to adapt to a carbon source limitation; however, four strains indicated the ability to secrete ligninolytic enzymes in all experimental conditions-Irpex lacteus, Pleurotus dryinus, Bjerkandera adusta, and Trametes versicolor-respectively displayed a 100%, 82.7%, 82.7%, and 55% oxidation of ABTS on lignin-containing media and 100%, 87.9%, 78%, and 70% oxidation of ABTS on hay-containing media after 168 h of incubation. As a result, the most potent strains of fungi were selected to produce lignocellulose-degrading enzymes and to demonstrate their potential application in biological lignocellulose pretreatment.

Dynamic protein deacetylation is a limited carbon source for acetyl-CoA–dependent metabolism

The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-CoA (AcCoA), which fuels essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% to 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability, and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent, ATP citrate lyase-deficient mouse embryonic fibroblasts (Acly-/- MEFs), and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly-/- MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand.

Characterization and removal mechanism of a novel enrofloxacin-degrading microorganism, Microbacterium proteolyticum GJEE142 capable of simultaneous removal of enrofloxacin, nitrogen and phosphorus

In the study, a novel ENR-degrading microorganism, Microbacterium proteolyticum GJEE142 was isolated from aquaculture wastewater for the first time. The ENR removal of strain GJEE142 was reliant upon the provision of limited additional carbon source, and was adaptative to low temperature (13 ℃) and high salinity (50‰). The ENR removal process, to which intracellular enzymes made more contributions, was implemented in three proposed pathways. During the removal process, oxidative stress response of strain GJEE142 was activated and the bacterial toxicity of ENR was decreased. Strain GJEE142 could also achieve the synchronous removal of ammonium, nitrite, nitrate and phosphorus with the nitrogen removal pathways of nitrate → nitrite → ammonium → glutamine → glutamate → glutamate metabolism and nitrate → nitrite → gaseous nitrogen. The phosphorus removal was implemented under complete aerobic conditions with the assistance of polyphosphate kinase and exopolyphosphatase. Genomic analysis provided corresponding genetic insights for deciphering removal mechanisms of ENR, nitrogen and phosphorus. ENR, nitrogen and phosphorus in both actual aquaculture wastewater and domestic wastewater could be desirably removed. Desirable adaptation, excellent performance and wide distribution will make strain GJEE142 the hopeful strain in wastewater treatment.

Carbon Sink Limitation Determines the Formation of the Altitudinal Upper Limit of an Evergreen Oak in Eastern China

Temperature is a critical environmental factor determining the upper limits of evergreen broadleaved tree taxa. However, whether carbon source or carbon sink limitation shapes this limit is not yet fully understood. We studied a subtropical evergreen oak (Cyclobalanopsis gracilis) at the northern limit of its distributional range. Along an elevational/temperature gradient towards its upper limit, we surveyed the variations in non-structural carbohydrate (NSC) concentrations of C. gracilis adults for 3 years. Additionally, a carbon balance manipulation experiment of debudding and defoliation was done to C. gracilis seedlings close to the upper distributional limit, aiming at investigating the changes in NSC concentrations and growth rates in different treatment groups. Our results showed that increasing elevation or decreasing temperature did not affect the trends of NSC concentration in twigs, old branches, or trunks of adults, nor did carbon balance manipulations (debudding or defoliation) of seedlings have a significant effect on the growth, while defoliation decreased NSC concentration in twigs. These results suggest that carbon sink limitation is the key physiological mechanism underlying low temperature in the shaping of this dominant evergreen broadleaved tree species in eastern China. Therefore, the formation of upper limits in evergreen oaks is most likely the result of a direct low-temperature restriction on meristematic activity and tissue formation instead of the result of insufficient carbon supply. More studies with expanded sample sizes are needed on other evergreen broadleaved tree species growing at their upper limits to confirm the carbon sink limitation hypothesis and reveal the detailed mechanisms.

Enhanced Nutrients Removal from Eutrophic Water by Aerobic Denitrifiers Assisting Floating Plants

Accelerated eutrophication is one of the most serious problems confronting freshwater systems worldwide. More effective methods to reduce nutrients from eutrophic water with high nitrogen but low carbon source are now urgently needed. In this study, the methods of floating plant remediation and aerobic denitrification were combined for seeking an effective strategy. Four aerobic denitrifying bacteria were adopted. Each strain was co-cultured with Eichhornia crassipes, and then the effects of different combinations on the removal of nitrogen and phosphorus were compared. Meanwhile, their impacts on the growth and physiological characteristics of plants were also measured. The results showed that the association of aerobic denitrifying bacteria with E. crassipes was effective in enhancing the simultaneous removal of nitrogen and phosphorus. E. crassipes helped bacteria to eliminate the accumulation of nitrites under the condition of limited carbon sources. Cooperation between Rhizobium rosettiformans and E. crassipes exhibited the best remediation performance. The four aerobic denitrifying bacteria increased the plant growth and phosphorus content, and had different effects on the plant root activity, leaf chlorophyll, leaf soluble sugar and leaf soluble protein concentration. Although the overall nutrients removal efficiency was greatly improved by the synergic association, the percentage contribution of plant uptake remained constant.

Developing a single-stage continuous process strategy for vitamin B12 production with Propionibacterium freudenreichii

BackgroundVitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. Besides, the use of Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) microorganisms, like Propionibacterium freudenreichii, especially non-GMO wild-type producers, are becoming an interesting alternative in markets where many final consumers have high health and ecological awareness. In this study, the production of vitamin B12 using the Propionibacterium freudenreichii NBRC 12391 wild-type strain was characterized and optimized in shake flasks before assessing several scale-up strategies.ResultsInitial results established that: (i) agitation during the early stages of the culture had an inhibitory effect on the volumetric production, (ii) 5,6-dimethylbenzimidazole (DMBI) addition was necessary for vitamin B12 production, and (iii) kinetics of vitamin B12 accumulation were dependent on the induction time when DMBI was added. When scaling up in a bioreactor, both batch and fed-batch bioprocesses proved unsuitable for obtaining high volumetric productivities mainly due to carbon source limitation and propionic acid inhibition, respectively. To overcome these drawbacks, an anaerobic single-phase continuous bioprocess strategy was developed. This culture strategy was maintained stable during more than 5 residence times in two independent cultures, resulting in 5.7-fold increase in terms of volumetric productivity compared to other scale-up strategies.ConclusionOverall, compared to previously reported strategies aimed to reduce propionic acid inhibition, a less complex anaerobic single-phase continuous and more scalable bioprocess was achieved.

Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa.

The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.

Does the repeated freezing and thawing of the aerobic layer affect the anaerobic release of N2O from SWIS?

Subsurface wastewater infiltration systems (SWIS) is an efficient, economical, and less temperature affected sewage treatment technology. Pollutants are removed by physical, chemical, and biological reactions such as filtration, adsorption, oxidation, and degradation. Under the conditions of limited carbon source and inactivation of nitrous oxide reductase, N2O, an important greenhouse gas, is released from the anaerobic layers of SWIS. However, is N2O release affected by repeated freeze-thawing of the upper aerobic layer? How does microbial population structure and denitrogenate activity in different profiles respond to the freeze-thaw cycle (FTC)? These questions have not yet been revealed. In this study, a SWIS simulator with in-situ regulation of FTC was first constructed. The re-distribution of N2O, microbial composition and denitrogenate activity were analyzed in response to FTC. Furthermore, potential bio-markers were screened and identified. The results revealed that the release of N2O was correlated with FTC, with the anaerobic layer being the main contributor throughout, accounting for 73.32–75.8 % of the total release. The limiting factor for N2O emissions was the NO3−-N concentration in the anaerobic zone, and there were no simple linear communications between total nitrogen and N2O generations. High throughput sequencing results showed the main markers of SWIS were Proteobacteria, Gemmatimonadetes, Firmicutes, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi and Nitrospirae, accounting for 97.4 %–98.1 % of the total relative abundance. A significant positive correlation between Firmicutes and anaerobic release of N2O was observed, where Firmicutes abundance increased from 5 % to 21 % during the experimental cycle, while N2O concentration increased from 2.65 mg·L−1 to 18.88 mg·L−1. The results indicated that Firmicutes was an important biomarker of N2O release under freeze-thaw conditions.

Establishment of low-carbon microbial network to enhance phenol removal in microbial electrochemical systems with whole process carbon source limitation

Microbial electrochemical system employs electroactive microorganisms as catalysts to generate energy and remove pollutants. The formation of electroactive biofilms (EABs) is essential to efficient pollutant removal as it provides the opportunity for microbial interspecies interaction. Bioavailable carbon source serves as the material basis for the energy supply, and its concentration plays a decisive role in EAB formation and pollutant removal. Unfortunately, the mechanisms of how bioavailable carbon source concentration affects microbial interspecies communication and metabolic activity during EAB formation and pollutant degradation have not been thoroughly investigated. Here, phenol was used as the pollutant to evaluate the performance of EABs formed at different acetate loadings (0.2, 0.5, and 1.0 g/L, marked as EAB-0.2 to 1.0). More phenol degrading microorganisms were enriched in EAB-0.2, achieving better phenol removal. Limited bioavailable carbon source forced microorganisms to adjust metabolites, thus facilitating the interspecies interaction. The close linkage between the up-regulated metabolic pathways facilitated the tricarboxylic acid cycle, leading to faster substrate metabolism. We demonstrated that EAB cultivated in a limited organic environment was more active in pollutant removal, which was essential to guide the application of MES in industrial wastewater treatment.