P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).
Although freshwater biomes cover less than 1% of the Earth's surface, they have disproportionate ecological significances. Attempts to study the taxonomy and function of freshwater microbiota are currently limited to samples collected from temperate lakes. In this study, we investigated samples from the photic and aphotic of an aquaculture site (disturbed) of Temengor Lake, a tropical lake in comparison with the undisturbed site of the lake using 16S rRNA amplicon and shotgun metagenomic approaches. Vertical changes in bacterial community composition and function of the Temengor Lake metagenomes were observed. The photic water layer of Temengor Lake was dominated by typical freshwater assemblages consisting of Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia, and Cyanobacteria lineages. On the other hand, the aphotic water featured in addition to Proteobacteria, Bacteroidetes, Verrucomicrobia, and two more abundant bacterial phyla that are typically ubiquitous in anoxic habitats (Chloroflexi and Firmicutes). The aphotic zone of Temengor Lake exhibited genetic potential for nitrogen and sulfur metabolisms for which terminal electron acceptors other than oxygen are used in the reactions. The aphotic water of the disturbed site also showed an overrepresentation of genes associated with the metabolism of carbohydrates, likely driven by the enrichment of nutrient resulting from aquaculture activities at the site. The results presented in this study can serve as a basis for understanding the structure and functional capacity of the microbial communities in the photic and aphotic zones/water layers of tropical man-made lakes.
Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.
In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
The effect of temperature on the efficiency of organics and nutrients removal during the cultivation of aerobic granular sludge (AGS) in biological treatment of synthetic wastewater was studied. With this aim, three 3 L sequencing batch reactors (SBRs) with influent loading rate of 1.6 COD g (L d)(-1) were operated at different high temperatures (30, 40 and 50 °C) for simultaneous COD, phosphate and ammonia removal at a complete cycle time of 3 h. The systems were successfully started up and progressed to steady state at different cultivation periods. The statistical comparison of COD, phosphate and ammonia for effluent from the three SBRs revealed that there was a significant difference between groups of all the working temperatures of the bioreactors. The AGS cultivated at different high temperatures also positively correlated with the accumulation of elements including carbon, oxygen, phosphorus, silicon, iron, aluminium, calcium and magnesium that played important roles in the granulation process.
In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.
The growth and lectin production of Ganoderma applanatum, a white rot fungus, was optimized in broth cultures. The fungus was found to have a higher growth rate and higher lectin activity when grown in a medium adjusted to pH 6.5 at 26°C under stationary conditions. Expression of lectin activity started in 5-day-old mycelial culture; maximum activity was expressed after the 15th day of incubation. Among the various carbon and nitrogen sources tested, the carbon source sucrose and the nitrogen source yeast extract support maximum growth and lectin production. Lectin from G. applanatum was purified by ammonium sulfate precipitation and ion exchange chromatography. The purified fraction revealed a single band with a molecular weight of 35.0 kDa. Moreover, carbohydrates such as mannitol, glucose, sucrose, maltose, mannose, galactose, sorbose, and fructose were found to inhibit the hemagglutinating activity of the lectin. The purified lectins from G. applanatum contain cytotoxic and proapoptotic activities against HT-29 colon adenocarcinoma cells.
Soil salinity exert negative impacts on agricultural production and regarded as a crucial issue in global wetland rice production (Oryza sativa L.). Indigenous salt-tolerant plant growth-promoting rhizobacteria (Bacillus sp.) could be used for improving rice productivity under salinity stress. This study screened potential salt-tolerant plant growth-promoting rhizobacteria (PGPR) collected from coastal salt-affected rice cultivation areas under laboratory and glasshouse conditions. Furthermore, the impacts of these PGPRs were tested on biochemical attributes and nutrient contents in various rice varieties under salt stress. The two most promising PGPR strains, i.e., 'UPMRB9' (Bacillus tequilensis 10b) and 'UPMRE6' (Bacillus aryabhattai B8W22) were selected for glasshouse trial. Results indicated that 'UPMRB9' improved osmoprotectant properties, i.e., proline and total soluble sugar (TSS), antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, 'UPMRB9' inoculated rice plants accumulated higher amount of nitrogen and calcium in tissues. Therefore, the indigenous salt-tolerant PGPR strain 'UPMRB9' could be used as a potential bio-augmentor for improving biochemical attributes and nutrient uptake in rice plants under salinity stress. This study could serve as a preliminary basis for future large-scale trials under glasshouse and field conditions.
Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.
Tropical peatlands significantly influence local and global carbon and nitrogen cycles, yet they face growing pressure from anthropogenic activities. Land use changes, such as peatland forests conversion to oil palm plantations, affect the soil microbiome and greenhouse gas (GHG) emissions. However, the temporal dynamics of microbial community changes and their role as GHG indicators are not well understood. This study examines the dynamics of peat chemistry, soil microbial communities, and GHG emissions from 2016 to 2020 in a logged-over secondary peat swamp forest in Sarawak, Malaysia, which transitioned to an oil palm plantation. This study focuses on changes in genetic composition governing plant litter degradation, methane (CH4), and nitrous oxide (N2O) fluxes. Soil CO2 emission increased (doubling from approximately 200 mg C m-2 h-1), while CH4 emissions decreased (from 200 µg C m-2 h-1 to slightly negative) following land use changes. The N2O emissions in the oil palm plantation reached approximately 1,510 µg N m-2 h-1, significantly higher than previous land uses. The CH4 fluxes were driven by groundwater table, humification levels, and C:N ratio, with Methanomicrobia populations dominating methanogenesis and Methylocystis as the main CH4 oxidizer. The N2O fluxes correlated with groundwater table, total nitrogen, and C:N ratio with dominant nirK-type denitrifiers (13-fold nir to nosZ) and a minor role by nitrification (a threefold increase in amoA) in the plantation. Proteobacteria and Acidobacteria encoding incomplete denitrification genes potentially impact N2O emissions. These findings highlighted complex interactions between microbial communities and environmental factors influencing GHG fluxes in altered tropical peatland ecosystems.IMPORTANCETropical peatlands are carbon-rich environments that release significant amounts of greenhouse gases when drained or disturbed. This study assesses the impact of land use change on a secondary tropical peat swamp forest site converted into an oil palm plantation. The transformation lowered groundwater levels and changed soil properties. Consequently, the oil palm plantation site released higher carbon dioxide and nitrous oxide compared to previous land uses. As microbial communities play crucial roles in carbon and nitrogen cycles, this study identified environmental factors associated with microbial diversity, including genes and specific microbial groups related to nitrous oxide and methane emissions. Understanding the factors driving microbial composition shifts and greenhouse gas emissions in tropical peatlands provides baseline information to potentially mitigate environmental consequences of land use change, leading to a broader impact on climate change mitigation efforts and proper land management practices.
A split plot 3 by 4 experiment was designed to characterize the relationship between production of gluthatione (GSH), oxidized gluthatione (GSSG), total flavonoid, anthocyanin, ascorbic acid and antioxidant activities (FRAP and DPPH) in three varieties of Labisia pumila Blume, namely the varieties alata, pumila and lanceolata, under four levels of nitrogen fertilization (0, 90, 180 and 270 kg N/ha) for 15 weeks. The treatment effects were solely contributed by nitrogen application; there was neither varietal nor interaction effects observed. As the nitrogen levels decreased from 270 to 0 kg N/ha, the production of GSH and GSSG, anthocyanin, total flavonoid and ascorbic acid increased steadily. At the highest nitrogen treatment level, L. pumila exhibited significantly lower antioxidant activities (DPPH and FRAP) than those exposed to limited nitrogen growing conditions. Significant positive correlation was obtained between antioxidant activities (DPPH and FRAP), total flavonoid, GSH, GSSG, anthocyanin and ascorbic acid suggesting that an increase in the antioxidative activities in L. pumila under low nitrogen fertilization could be attributed to higher contents of these compounds. From this observation, it could be concluded that in order to avoid negative effects on the quality of L. pumila, it is advisable to avoid excessive application of nitrogen fertilizer when cultivating the herb for its medicinal use.
A split plot 3 by 4 experiment was designed to examine the impact of 15-week variable levels of nitrogen fertilization (0, 90, 180 and 270 kg N/ha) on the characteristics of total flavonoids (TF), total phenolics (TP), total non structurable carbohydrate (TNC), net assimilation rate, leaf chlorophyll content, carbon to nitrogen ratio (C/N), phenyl alanine lyase activity (PAL) and protein content, and their relationships, in three varieties of Labisia pumila Blume (alata, pumila and lanceolata). The treatment effects were solely contributed by nitrogen application; there was neither varietal nor interaction effect observed. As nitrogen levels increased from 0 to 270 kg N/ha, the production of TNC was found to decrease steadily. Production of TF and TP reached their peaks under 0 followed by 90, 180 and 270 kg N/ha treatment. However, net assimilation rate was enhanced as nitrogen fertilization increased from 0 to 270 kg N/ha. The increase in production of TP and TF under low nitrogen levels (0 and 90 kg N/ha) was found to be correlated with enhanced PAL activity. The enhancement in PAL activity was followed by reduction in production of soluble protein under low nitrogen fertilization indicating more availability of amino acid phenyl alanine (phe) under low nitrogen content that stimulate the production of carbon based secondary metabolites (CBSM). The latter was manifested by high C/N ratio in L. pumila plants.
Polyhydroxyalkanoates (PHAs) has been paid great attention because of its useful thermoplastic properties and complete degradation in various natural environments. But, at industrial level, the successful commercialization of PHAs is limited by the high production cost due to the expensive carbon source and recovery processes. Pseudomonas mendocina PSU cultured for 72 h in mineral salts medium (MSM) containing 2% (v/v) biodiesel liquid waste (BLW) produced 79.7 wt% poly(3-hydroxybutyrate) (PHB) at 72 h. In addition, this strain produced 43.6 wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 8.6 HV mol% at 60 h when added with 0.3% sodium propionate. The synthesized intracellular PHA granules were recovered and purified by the recently reported biological method using mealworms. The weight average molecular weight (Mw ) and number average molecular weight (Mn ) of the biologically extracted PHA were higher than that from the chloroform extraction with comparable melting temperature (Tm ) and high purity. This study has successfully established a low-cost process to synthesize PHAs from BLW and subsequently confirmed the ability of mealworms to extract PHAs from various kinds of bacterial cells.