Heterotrophic Growth Conditions Research Articles

Selection and characterization of a Parachlorella kessleri microalgal strain able to assimilate lactose, and grow on dairy waste

One of the main limitations of commercial production of algae is the cost of cultivation, largely attributed to the cost of the nutrients; hence, finding cheap alternative substrates has been a significant line of research in this field. The dairy industry produces large amounts of wastewater that might be used as a cost-effective nutrient alternative, containing lactose as a main carbon source, as well as other essential nutrients like nitrogen and phosphate. Nevertheless, just a few algal species of commercial value can grow on any organic carbon sources, and even less are able to utilize lactose. In this work we have identified a Parachlorella kessleri strain capable of utilizing lactose for growth, and have characterized its ability to accumulate metabolites of commercial interest under heterotrophic growth conditions. P. kessleri was capable to utilize lactose from dairy wastewater, and to accumulate several amino acids, dicarboxylic acids, such as tartaric acid and succinic acid, and triacylglycerols in heterotrophic conditions. These metabolites have applications in the food, feed, and pharmaceutical industries, and in the green chemical industry for the production of bio-based green polymers and biofuels. The significance of these findings for future product development is discussed.

Cultivation of a Novel Strain of Chlorella vulgaris S2 under Phototrophic, Mixotrophic, and Heterotrophic Conditions, and Effects on Biomass Growth and Composition

Microalgal biomass is an excellent platform for producing food, feed, nutraceuticals, pharmaceuticals, and biofuels. This study aimed to investigate the effect of the trophic mode of cultivation (phototrophic, heterotrophic, and mixotrophic) on the growth and biomass composition of Chlorella vulgaris S2. The contents of lipids and carbohydrates, as well as the fatty acid composition of total lipids, were studied. The effects of the carbon-to-nitrogen ratio (C:N) and the organic carbon concentration of the growth media under mixotrophic and heterotrophic conditions were also investigated. The C:N ratio of 30 mol mol−1 favoured lipid synthesis, and the C:N ratio of 10 mol mol−1 favoured carbohydrate synthesis. Maximal lipid and biomass productivities (2.238 and 0.458 g L−1 d−1, respectively) were obtained under mixotrophic conditions at the C:N ratio of 50 mol mol−1 and glucose concentration of 50 g L−1. Fed-batch cultivation conducted in a stirrer tank bioreactor under heterotrophic growth conditions increased biomass (2.385 g L−1 d−1, respectively) and lipid (0.339 L−1 d−1) productivities ~50 and ~60 times compared to the fed-batch phototrophic cultivation, respectively. The trophic mode, growth phase, and growth medium composition significantly influenced the fatty acid composition. Under mixotrophic and heterotrophic growth conditions, lipid accumulation is associated with an increase in oleic acid (C18:1) content. Mixotrophically grown biomass of Chlorella vulgaris S2 under optimised conditions is a suitable source of lipids for biodiesel production.

Effect of macronutrients (carbon, nitrogen, and phosphorus) on the growth of Chlamydomonas reinhardtii and nutrient recovery under different trophic conditions.

More stringent discharge standards have led to the development of an alternative nutrient recovery system from wastewater. Microalgae cultivation in wastewater treatment works has presented considerable promise from the perspective of sustainable resource management. Growth kinetics models are useful tools to optimize nutrient recovery from wastewater by algal uptake. Therefore, this research aims to identify the growth kinetics of Chlamydomonas reinhardtii under both heterotrophic and phototrophic conditions with different nutrient concentrations that typify those found in wastewater treatment works. In addition, the effects of macronutrients (C, N, and P) on heterotrophic and phototrophic microalgae growth and nutrient recovery were studied. Greater specific growth rates were achieved under heterotrophic conditions than in phototrophic cultivation. The maximum specific growth rates and nutrient recovery efficiencies were achieved at 5 mg P L-1 under both heterotrophic and phototrophic growth conditions. Nitrate was the preferred form of nitrogen source under heterotrophic conditions, while nitrogen sources did not present any significant influences in the phototrophic cultivation. Specific growth rates reported for both heterotrophic and phototrophic microalgae at lower carbon concentrations (3.10 d-1 and 0.46 d-1, sequentially) were higher than those at higher carbon concentrations (1.95 d-1 and 0.22 d-1, respectively). C. reinhardtii presented an extreme capacity to adapt and grow at all experimental conditions tested in heterotrophic and phototrophic cultivations.

Rapid Enrichment of Cupriavidus necator in Mixed Microbial Cultures Using Autotrophic Growth: Mixed Microbial Cultures for Biodegradable Polymer Production Using CO2 and Organic Wastes

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that can replace petroleum-based plastics. Cupriavidus necator has been extensively utilized in industrial PHA production because the bacterium can grow and accumulate high amounts of PHA in their cells under heterotrophic and/or autotrophic growth conditions. Microbial PHA production using mixed cultures reduces the high PHA production costs and allows for the utilization of waste organics as carbon sources. However, C. necator cultivation in mixed microbial cultures has not been investigated before. In this work, we developed innovative methods for rapid and selective enrichment of C. necator in mixed cultures, utilizing the unique metabolic characteristics of C. necator (i.e., the ability to grow heterotrophically and/or autotrophically). Three enrichment methods were proposed: autotrophic-only, alternating autotrophic–heterotrophic, and heterotrophic-only cycles. These methods were examined with two wastewater sources as initial mixed mixtures. In the autotrophic-only method, the fraction of C. necator exceeded 92% of the total microbial population in only 60 h, whereas the alternating autotrophic–heterotrophic cycles showed >81.3% selection of C. necator. However, the heterotrophic-only method resulted in practically no enrichment of C. necator after five repeated feast–famine cycles. When tested for PHA accumulation, enriched cultures using the autotrophic and alternating cycles accumulated significant amounts of PHA, confirming the selective and rapid enrichment of C. necator over other microorganisms. Based on the experimental results, the autotrophic-based enrichment is a potential approach for the cost-effective cultivation of C. necator, which can reduce PHA production costs.

Unchanged nitrate and nitrite isotope fractionation during heterotrophic and Fe(II)-mixotrophic denitrification suggest a non-enzymatic link between denitrification and Fe(II) oxidation

Natural-abundance measurements of nitrate and nitrite (NOx) isotope ratios (δ15N and δ18O) can be a valuable tool to study the biogeochemical fate of NOx species in the environment. A prerequisite for using NOx isotopes in this regard is an understanding of the mechanistic details of isotope fractionation (15ε, 18ε) associated with the biotic and abiotic NOx transformation processes involved (e.g., denitrification). However, possible impacts on isotope fractionation resulting from changing growth conditions during denitrification, different carbon substrates, or simply the presence of compounds that may be involved in NOx reduction as co-substrates [e.g., Fe(II)] remain uncertain. Here we investigated whether the type of organic substrate, i.e., short-chained organic acids, and the presence/absence of Fe(II) (mixotrophic vs. heterotrophic growth conditions) affect N and O isotope fractionation dynamics during nitrate (NO3–) and nitrite (NO2–) reduction in laboratory experiments with three strains of putative nitrate-dependent Fe(II)-oxidizing bacteria and one canonical denitrifier. Our results revealed that 15ε and 18ε values obtained for heterotrophic (15ε-NO3–: 17.6 ± 2.8‰, 18ε-NO3–:18.1 ± 2.5‰; 15ε-NO2–: 14.4 ± 3.2‰) vs. mixotrophic (15ε-NO3–: 20.2 ± 1.4‰, 18ε-NO3–: 19.5 ± 1.5‰; 15ε-NO2–: 16.1 ± 1.4‰) growth conditions are very similar and fall within the range previously reported for classical heterotrophic denitrification. Moreover, availability of different short-chain organic acids (succinate vs. acetate), while slightly affecting the NOx reduction dynamics, did not produce distinct differences in N and O isotope effects. N isotope fractionation in abiotic controls, although exhibiting fluctuating results, even expressed transient inverse isotope dynamics (15ε-NO2–: –12.4 ± 1.3 ‰). These findings imply that neither the mechanisms ordaining cellular uptake of short-chain organic acids nor the presence of Fe(II) seem to systematically impact the overall N and O isotope effect during NOx reduction. The similar isotope effects detected during mixotrophic and heterotrophic NOx reduction, as well as the results obtained from the abiotic controls, may not only imply that the enzymatic control of NOx reduction in putative NDFeOx bacteria is decoupled from Fe(II) oxidation, but also that Fe(II) oxidation is indirectly driven by biologically (i.e., via organic compounds) or abiotically (catalysis via reactive surfaces) mediated processes co-occurring during heterotrophic denitrification.

Metabolic plasticity of mixotrophic algae is key for their persistence in browning environments

Light availability is the main regulator of primary production, shaping photosynthetic communities and their production of ecologically important biomolecules. In freshwater ecosystems, increasing dissolved organic carbon (DOC) concentrations, commonly known as browning, leads to lower light availability and the proliferation of mixotrophic phytoplankton. Here, a mixotrophic algal species (Cryptomonas sp.) was grown under five increasing DOC concentrations to uncover the plastic responses behind the success of mixotrophs in browning environments and their effect in the availability of nutritionally important biomolecules. In addition to the browning treatments, phototrophic, heterotrophic and mixotrophic growth conditions were used as controls. Despite reduced light availability, browning did not impair algal growth compared to phototrophic conditions. Comparative transcriptomics showed that genes related to photosynthesis were down‐regulated, whereas phagotrophy gene categories (phagosome, lysosome and endocytosis) were up‐regulated along the browning gradient. Stable isotope analysis of phospholipid fractions validated these results, highlighting that the studied mixotroph increases its reliance on heterotrophic processes with browning. Metabolic pathway reconstruction using transcriptomic data suggests that organic carbon is acquired through phagotrophy and used to provide energy in conjunction with photosynthesis. Although metabolic responses to browning were observed, essential fatty acid content was similar between treatments while sterol content was slightly higher upon browning. Together, our results provide a mechanistic model of how a mixotrophic alga responds to browning and how such responses affect the availability of nutritionally essential biomolecules for higher trophic levels.

Inhibition Effects of Free Ammonia (FA) on the Rates of Growth, Photosynthesis and Respiration of Chlorella vulgaris

The potential use of microalgae in advanced biological wastewater treatment processes has been actively studied due to their excellent nitrogen and phosphorus removal capabilities. This study investigated the inhibitory effects of free ammonia (FA) on microalgae under autotrophic, heterotrophic, and mixotrophic growth conditions, since the inhibitory effects in heterotrophic and mixotrophic modes has not been reported yet. Chlorella vulgaris were exposed to 3–20 mg-N L−1 of FA in the autotrophic, heterotrophic, and mixotrophic modes and the effects on growth, photosynthesis, and respiration were studied. The microalgal growth rate was 2–3 times greater in the mixotrophic mode than the other modes. Microalgal growth in the autotrophic mode was most vulnerable to FA and the growth inhibition was observed at FA concentration of ≥ 9 mg-N L−1. Similarly, the photosynthesis and respiration of C. vulgaris were inhibited at FA concentrations higher than 12 mg-N LT−1. The removal of carbon and ammonia was greater in the mixotrophic mode than the other modes. The ammonia removal by microalgae increased in proportion to the FA concentration unlike the carbon removal. This was attributed to the ammonium accumulation in microalgae due to the proton imbalance and differences in intracellular and external pH conditions, which were caused by the presence of excess FA in water. Overall, the results show that the mixotrophic microalgae have greater tolerance to FA, and hence, greater potential for use in treatment of wastewaters having high FA concentrations.

Establishing Mixotrophic Growth of Cupriavidus necator H16 on CO2 and Volatile Fatty Acids

The facultative chemolithoautotroph Cupriavidus necator H16 is able to grow aerobically either with organic substrates or H2 and CO2 s and it can accumulate large amounts of (up to 90%) poly (3-hydroxybutyrate), a polyhydroxyalkanoate (PHA) biopolymer. The ability of this organism to co-utilize volatile fatty acids (VFAs) and CO2 as sources of carbon under mixotrophic growth conditions was investigated and PHA production was monitored. PHA accumulation was assessed under aerobic conditions, with either individual VFAs or in mixtures, under three different conditions—with CO2 as additional carbon source, without CO2 and with CO2 and H2 as additional sources of carbon and energy. VFAs utilisation rates were slower in the presence of CO2. PHA production was significantly higher when cultures were grown mixotrophically and with H2 as an additional energy source compared to heterotrophic or mixotrophic growth conditions, without H2. Furthermore, a two-step VFA feeding regime was found to be the most effective method for PHA accumulation. It was used for PHA production mixotrophically using CO2, H2 and VFA mixture derived from an anaerobic digestor (AD). The data obtained demonstrated that process parameters need to be carefully monitored to avoid VFA toxicity and low product accumulation.

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.

Glucose-assisted trophic conversion of Chlamydomonas reinhardtii by expression of glucose transporter GLUT1

The use of genetic engineering techniques to alter the nutritional patterns of microalgae is a key means of enhancing microalgal biomass, as it enables the highly efficient heterotrophic growth of microalgae via the utilisation of organic carbon sources, especially glucose. To investigate the effects of the expression of the glucose transporter on the growth of the model alga, Chlamydomonas reinhardtii, we transformed C. reinhardtii using the human GLUT1 gene, regulated by the Hsp70, PsaD, and Arg7 promoters, and obtained engineered algae after screening. The engineered algae showed better growth than the control algae under both heterotrophic and mixotrophic growth conditions. The engineered alga in which the GLUT1 gene was regulated by the Arg7 promoter exhibited the best growth. Although the engineered algae exhibited heterotrophic growth in complete darkness by using glucose as the carbon source, their growth rates and biomass were relatively low compared to those of the control. Analysis of the glucose transport ability of the engineered algae revealed that glucose was successfully transported to the cytoplasm via the glucose transporters, but was unable to enter the chloroplasts, which could explain the slow heterotrophic growth of the engineered algae.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Antimicrobial Potential of Microorganisms Isolated from the Bottom Sediments of Lake Baikal.

Extremophilic microorganisms attract researchers by their unique characteristics, primarily antagonistic ones, acquired in the process of survival in extreme natural conditions. The antimicrobial potential of the metabolites of these microorganisms is quite broad, from the food industry to therapeutic drugs. Microbial mats of Lake Baikal are a source of unique and diverse microorganisms. The study aimed to evaluate the antimicrobial activity of bacterial strains isolated from the bottom sediments of the lake. Using heterotrophic growth conditions, seven bacterial strains were isolated from samples collected in several coastal zones of Lake Baikal. Thisstudy identified both widespread strains of the genera Pseudomonas and Bacillus and rare genera Micrococcus and Acinetobacterrepresentatives. Metabolites of five strains were found to have a broad spectrum of antimicrobial activity. Four large fractions of metabolites of the isolated strains wereidentified. Two peptides of the isolated fractions of metabolites (one is produced by microorganisms of all five isolated strains, another—only by Pseudomonas putida) are low molecular weight oligopeptides. These peptides were proved to be bacteriocins.

Genome-Scale Analysis of Acetobacterium woodii Identifies Translational Regulation of Acetogenesis.

ABSTRACTAcetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity.IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

Metabolomic Study of Heterotrophically Grown Chlorella sp. Isolated from Wastewater in Northern Sweden.

There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga’s lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health.

Model based analysis of carbon fluxes within microalgae-bacteria flocs using respirometric-titrimetric data

With the emerging need of nutrient recycling in resource recovery facilities, the use of microalgae-bacteria flocs has received considerable attention in the past few years. However, although the main biological processes are already known, the complex interactions occurring between algae and bacteria are not fully understood. In this work, a combined respirometric-titrimetric unit was used to assess the microorganisms' kinetics within microalgae-bacteria flocs under different growth regimes (i.e. photoautotrophic, heterotrophic and mixotrophic) and different ratios of inorganic (IC) to organic carbon (OC) (IC:OC-ratios). Using this respirometric-titrimetric data, a new model was developed, calibrated and successfully validated. The model takes into account the heterotrophic growth of bacteria, the photoautotrophic, heterotrophic and mixotrophic growth of algae and the production and consumption of extracellular polymeric substances (EPS) by both bacteria and algae. As such, the model can be used for detailed analysis of the carbon fluxes within microalgae-bacteria flocs in an efficient way. Model analysis revealed the high importance of the EPS regulatory mechanism. Firstly, under heterotrophic growth conditions, OC-uptake occurred during the first 10–15 min. This was linked with internal OC storage (49% of added OC) and EPS production (40%), as such providing carbon reserves which can be consumed during famine conditions. Moreover, the algae were able to compete with bacteria for OC. Secondly, under photoautotrophic conditions, algae used the added IC to grow (57% of added IC) and to produce EPS (29%), which consecutively stimulated heterotrophic bacteria growth (20%). Finally, under mixotrophic conditions, low IC:OC-ratios resulted in an extensive OC-storage and EPS production (50% of added C) and an enhanced microalgal CO2 reuse, resulting in an increased algal growth of up to 29%.

Evaluation of Desmodesmus subspicatus for the treatment of wastewater

ABSTRACT Microalgae are diverse in nature, capable to survive under diverse environmental conditions, and a good method of choice for remediation of polluted waters. Desmodesmus is a recently updated genus having capacity to consume wastewater nutrients under heterotrophic growth conditions. The aim of this study is to investigate the potential of Desmodesmus subspicatus for the treatment of wastewater. The treatment of wastewater was carried out for the period of 20 days at ambient temperature under continuous (24 hr) illumination of 4750 lm by a LED source withaeration at rate of 3.5 L/min by air pump. Treated wastewater samples with microalgae were then analysed for pH, total dissolved solids (TDS), biological oxygen demand (BOD), chemical oxygen demand (COD), ammonia as NH4 + phosphate and nitrate on every 5th day during experiment to monitor the remediation process. The results showed that D. subspicatus significantly lowered COD (84%) and BOD (76%) levels. The removal efficiency of ammonia was maximum (approximately 100%), nitrate (91%), and phosphate (71%) from wastewater was also prominently observable. The treatment effectively reduced (80%) TDS up to permissible limits. D. subspicatus can growin nutrient rich waters and has the potential of remediation under photoautotrophic conditions.

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

PDM4, a Pentatricopeptide Repeat Protein, Affects Chloroplast Gene Expression and Chloroplast Development in Arabidopsis thaliana.

Extensive studies have been carried out on chloroplast gene expression and chloroplast development; however, the regulatory mechanism is still largely unknown. Here, we characterized Pigment-Defective Mutant4 (PDM4), a P-type PPR protein localized in chloroplast. The pdm4 mutant showed seedling-lethal and albino phenotype under heterotrophic growth conditions. Transmission electron microscopic analysis revealed that thylakoid structure was totally disrupted in pdm4 mutant and eventually led to the breakdown of chloroplasts. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in pdm4 mutant. Besides, transcript profile analysis detected that, in pdm4 mutant, the expression of plastid-encoded RNA polymerase-dependent genes was markedly affected, and deviant chloroplast rRNA processing was also observed. In addition, we found that PDM4 functions in the splicing of group II introns and may also be involved in the assembly of the 50S ribosomal particle. Our results demonstrate that PDM4 plays an important role in chloroplast gene expression and chloroplast development in Arabidopsis.

Systems biology approach identifies functional modules and regulatory hubs related to secondary metabolites accumulation after transition from autotrophic to heterotrophic growth condition in microalgae.

Heterotrophic growth mode is among the most promising strategies put forth to overcome the low biomass and secondary metabolites productivity challenge. To shedding light on the underlying molecular mechanisms, transcriptome meta-analysis was integrated with weighted gene co-expression network analysis (WGCNA), connectivity analysis, functional enrichment, and hubs identification. Meta-analysis and Functional enrichment analysis demonstrated that most of the biological processes are up-regulated at heterotrophic growth condition, which leads to change of genetic architectures and phenotypic outcomes. WGNCA analysis of meta-genes also resulted four significant functional modules across logarithmic (LG), transition (TR), and production peak (PR) phases. The expression pattern and connectivity characteristics of the brown module as a non-preserved module vary across LG, TR, and PR phases. Functional analysis identified Carotenoid biosynthesis, Fatty acid metabolism and Methane metabolism as enriched pathways in the non-preserved module. Our integrated approach was applied here, identified some hubs, such as a serine hydroxymethyltransferase (SHMT1), which is the best candidate for development of metabolites accumulating strains in microalgae. Current study provided a new insight into underlying metabolite accumulation mechanisms and opens new avenue for the future applied studies in the microalgae field.

Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.