Simultaneous dairy wastewater treatment and bioelectricity production in a new microbial fuel cell using photosynthetic Synechococcus / Tratamiento simultáneo de aguas residuales lácteas y producción de bioelectricidad en una nueva celda de combustible microbiana utilizando Synechococcus fotosintético

Photosynthetic microbial fuel cell (PMFC) is a novel technology, which employs organic pollutants and organisms to produce electrons and biomass and capture CO2 by bio-reactions. In this study, a new PMFC was developed based on Synechococcus sp. as a biocathode, and dairy wastewater was used in the anode chamber. Different experiments including batch feed mode, semi-continuous feed mode, Synechococcus feedstock to the anode chamber, Synechococcus-Chlorella mixed system, the feedstock of treated wastewater to the cathode chamber, and use of extra nutrients in the anodic chamber were performed to investigate the behavior of the PMFC system. The results indicated that the PMFC with a semi-continuous feed mode is more effective than a batch mode for electricity generation and pollutant removal. Herein, maximum power density, chemical oxygen demand removal, and Coulombic efficiency were 6.95 mW/m2 (450 Ω internal resistance), 62.94, and 43.16%, respectively, through mixing Synechococcus sp. and Chlorella algae in the batch-fed mode. The maximum nitrate and orthophosphate removal rates were 98.83 and 68.5%, respectively, wherein treated wastewater in the anode was added to the cathode. No significant difference in Synechococcus growth rate was found between the cathodic chamber of PMFC and the control cultivation cell. The heating value of the biocathode biomass at maximum Synechococcus growth rate (adding glucose into the anode chamber) was 0.2235 MJ/Kg, indicating the cell’s high ability for carbon dioxide recovery. This study investigated not only simultaneous bioelectricity production and dairy wastewater in a new PMFC using Synechococcus sp. but also studied several operational parameters and presented useful information about their effect on PMFC performance.(AU)

A symbiotic yeast to enhance heterotrophic and mixotrophic cultivation of Chlorella pyrenoidosa using sucrose as the carbon source.

Heterotrophic or mixotrophic culture of microalgae is feasible alternative approach to avoid light limitation in autotrophic culture. However, only a few kinds of organic carbon sources are available for algal culture. Disaccharides, such as sucrose, are difficult to be utilized by microalgae under both heterotrophic and mixotrophic conditions. In this study, a symbiotic yeast was accidentally found in a contaminated algal suspension. The symbiotic yeast was isolated and identified as Cryptococcus sp. This yeast was able to extracellularly hydrolyze sucrose and accumulated monosaccharides in the medium. It can enhance algal growth using sucrose as the carbon source at both heterotrophic and mixotrophic modes when mix-cultured with Chlorella pyrenoidosa. The highest algal cell density of 118.8 × 106 and 151.2 × 106 cells/mL was achieved with a final algal percentage of 83.5 and 93.2% at heterotrophic and mixotrophic culture, respectively. Furthermore, the protein and lipid content was significantly enhanced by mix-culture C. pyrenoidosa with Cryptococcus YZU-1. The fatty acid accumulated in this co-culture system was suitable for the production of biodiesel. This symbiotic yeast solved the problem that C. pyrenoidosa cannot heterotrophically or mixotrophically utilize sucrose. A high algae density was obtained and the protein and lipid accumulation were also significantly enhanced. This study provided a novel approach for production of protein or lipid-rich biomass using sucrose or sucrose-rich wastes as the carbon source.

Comparative assessment of pretreatment strategies for production of microalgae-based biodiesel from locally isolated Chlorella homosphaera.

The yield and quality of lipids extracted from microalgal biomass are critical factors in the production of microalgae-based biodiesel. The green microalga Chlorella homosphaera, isolated from Beira Lake, Colombo, Sri Lanka was employed in the present study to identify the effect of chlorophyll removal and cell disruption methods on lipid extraction yield, fatty acid methyl ester (FAME) profile and quality parameters of biodiesel; including cetane number (CN), iodine value (IV), degree of unsaturation (DU) and high heating value (HHV). In the first section of this study, chlorophyll was removed from dry microalgae biomass prior to lipid extraction. Through the analysis of FAME profiles, it was observed that chlorophyll removal yielded biodiesel of enhanced quality, albeit with a lipid loss of 44.2% relative to the control. In the second section of the study, mechanical cell disruption strategies including grinding, autoclaving, water bath heating and microwaving were employed to identify the most effective method to improve lipid recovery from chlorophyll-removed microalgae biomass. Autoclaving (121 °C, 20 min sterilization time, total time 2 h) was the most effective cell disruption technique of the methods tested, in terms of lipid extraction yield (39.80%) and also biodiesel quality. Moreover, it was observed that employing cell disruption subsequent to chlorophyll removal has a significant impact on the FAME profile of microalgae-based biodiesel, and consequently served to increase HHV and CN although IV and DU did not vary significantly.

d-Lactic acid secreted by Chlorella fusca primes pattern-triggered immunity against Pseudomonas syringae in Arabidopsis.

Biological control agents including microbes and their products have been studied as sustainable crop protection strategies. Although aquatic microalgae have been recently introduced as a biological control agent, the underlying molecular mechanisms are largely unknown. The aim of the present study was to investigate the molecular mechanisms underlying biological control by microalga Chlorella fusca. Foliar application of C. fusca elicits induced resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 that activates plant immunity rather than direct antagonism. To understand the basis of C. fusca-triggered induced resistance at the transcriptional level, we conducted RNA sequencing (RNA-seq) analysis. RNA-seq data showed that, upon pathogen inoculation, C. fusca treatment primed the expression of cysteine-rich receptor-like kinases, WRKY transcription factor genes, and salicylic acid and jasmonic acid signalling-related genes. Intriguingly, the application of C. fusca primed pathogen-associated molecular pattern -triggered immunity, characterized by reactive oxygen species burst and callose deposition, upon flagellin 22 treatment. The attempts to find C. fusca determinants allowed us to identify d-lactic acid secreted in the supernatant of C. fusca as a defence priming agent. This is the first report of the mechanism of innate immune activation by aquatic microalga Chlorella in higher plants.

Genetic basis for the establishment of endosymbiosis in Paramecium.

The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria. The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria, and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

Increasing the autotrophic growth of Chlorella USTB-01 via the control of bacterial contamination by Bdellovibrio USTB-06.

AIMS: (i) To obtain and identify the predatory bacteria for the control of contaminated bacteria and to promote the autotrophic growth of Chlorella USTB-01. (ii) To identify and measure the different cell numbers in microalgal culture using flow cytometer. METHODS AND RESULTS: A predatory bacterial strain was isolated using Escherichia coli BL21 as a sole prey host, which was identified as Bdellovibrio USTB-06 by the analysis of 16S rDNA sequence. A flow cytometer was successfully used to identify and measure the cell numbers of Chlorella USTB-01, the contaminated bacteria and Bdellovibrio USTB-06 simultaneously in the autotrophic culture of Chlorella USTB-01 according to the identification of the different cell sizes. With the addition of Bdellovibrio USTB-06 at initial 104 plaque-forming units per ml, the contaminated bacteria severely decreased by about five counts (in log10  CFU per ml) and the growth of Chlorella USTB-01 was greatly increased by 37·0% compared with those of control respectively. CONCLUSIONS: Bdellovibrio USTB-06 could effectively promote the growth of Chlorella USTB-01 via the killing of the contaminated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals a good biotechnology method to increase the growth of Chlorella USTB-01 which is very important in the industry of microalgal culture.

Antimicrobial-resistant Faecal Organisms in Algae Products Marketed as Health Supplements.

Dietary supplements are increasingly popular in Irish society. One of these is blue-green algae which is used with a variety health benefits in mind. A batch of Chlorella powder was found to be contaminated with Salmonella species in Ireland in 2015. This prompted additional testing of a total of 8 samples of three different products (Chlorella, Spirulina and Super Greens), for other faecal flora and antimicrobial resistance in any bacteria isolated. All 8 samples cultured enteric flora such as Enterococci, Enterobacteriaceae and Clostridium species. Antimicrobial susceptibility testing revealed one isolate with extended-spectrum ?-lactamase (ESBL) activity and one with carbapenemase activity. Clinicians caring for vulnerable patients should be aware of the potential risk of exposure to antimicrobial resistant bacteria associated with these products.

Microbial communities of biomethanization digesters fed with raw and heat pre-treated microalgae biomasses.

Microalgae biomasses are considered promising feedstocks for biofuel and methane productions. Two Continuously Stirred Tank Reactors (CSTR), fed with fresh (CSTR-C) and heat pre-treated (CSTR-T) Chlorella biomass were run in parallel in order to determine methane productions. The methane yield was 1.5 times higher in CSTR-T with regard to CSTR-C. Aiming to understand the microorganism roles within of the reactors, the sludge used as an inoculum (I), plus raw (CSTR-C) and heat pre-treated (CSTR-T) samples were analyzed by high-throughput pyrosequencing. The bacterial communities were dominated by Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes. Spirochaetae and Actinobacteria were only detected in sample I. Proteobacteria, mainly Alfaproteobacteria, were by far the dominant phylum within of the CSTR-C bioreactor. Many of the sequences retrieved were related to bacteria present in activated sludge treatment plants and they were absent after thermal pre-treatment. Most of the sequences affiliated to the Bacteroidetes were related to uncultured groups. Anaerolineaceae was the sole family found of the Chloroflexi phylum. All of the genera identified of the Firmicutes phylum carried out macromolecule hydrolysis and by-product fermentation. The proteolytic bacteria were prevalent over the saccharolytic microbes. The percentage of the proteolytic genera increased from the inoculum to the CSTR-T sample in a parallel fashion with an available protein increase owing to the high protein content of Chlorella. To relate the taxa identified by high-throughput sequencing to their functional roles remains a future challenge.

Co-generation of biohydrogen and biomethane through two-stage batch co-fermentation of macro- and micro-algal biomass.

Aquatic micro-algae can be used as feedstocks for gaseous biofuel production via biological fermentation. However, micro-algae usually have low C/N ratios, which are not advantageous for fermentation. In this study, carbon-rich macro-algae (Laminaria digitata) mixed with nitrogen-rich micro-algae (Chlorella pyrenoidosa and Nannochloropsis oceanica) were used to maintain a suitable C/N ratio of 20 for a two-stage process combining hydrogen and methane fermentation. Co-fermentation of L. digitata and micro-algae facilitated hydrolysis and acidogenesis, resulting in hydrogen yields of 94.5-97.0mL/gVS; these values were 15.5-18.5% higher than mono-fermentation using L. digitata. Through the second stage of methane co-fermentation, a large portion of energy remaining in the hydrogenogenic effluents was recovered in the form of biomethane. The two-stage batch co-fermentation markedly increased the energy conversion efficiencies (ECEs) from 4.6-6.6% during the hydrogen fermentation to 57.0-70.9% in the combined hydrogen and methane production.

[Effects of a Symbiotic Bacterium on the Accumulation and Transformation of Arsenate by Chlorella salina].

Algae-bacteria consortia may be potentially applied in wastewater treatment and environment remediation. In this study, in order to investigate effects of a symbiotic bacterium on the accumulation and transformation of arsenate[As(Ⅴ)] by Chlorella salina, we used batch cultures to determine the uptake, adsorption and transformation of As by axenic and non-axenic C. salina exposed to 0-750 µg·L-1 As(Ⅴ) for 7 d. The symbiotic bacterium of C. salina was confirmed to be Halomonas sp. after isolation, cultivation and 16S rRNA identification. The bacterial presence markedly increased the adsorption of As in C. salina, but it markedly reduced the absorption and the toxic effect of As(Ⅴ). Arsenate was the major arsenic species in the cells of axenic and non-axenic C. salina. The proportion of arsenite[As(Ⅲ)] was 8.99%-11.52% in the axenic microalga whereas a small quantity of monomethylarsonous acid (MMA) and dimethylarsinous acid (DMA) (0.02%-0.04%) were detected in the non-axenic counterpart. As(Ⅲ) dominated the As speciation in the bacterial culture and the percentage of As(Ⅴ) was 7.59%-26.80%, indicating that this symbiotic bacterium had a strong As(Ⅴ) reducing ability. The As removal rate (19.81%-41.08%) by non-axenic C. salina was higher than the bacterium alone (5.14%-14.62%) and axenic C. salina (14.98%-21.08%) after 7 d As(Ⅴ) exposure. The symbiotic Halomonas sp. promoted the accumulation of As by C. salina, indicating that algae-bacteria consortia might enhance the bioremediation of As contaminated water.

A diverse assemblage of indole-3-acetic acid producing bacteria associate with unicellular green algae.

Microalgae have tremendous potential as a renewable feedstock for the production of liquid transportation fuels. In natural waters, the importance of physical associations and biochemical interactions between microalgae and bacteria is generally well appreciated, but the significance of these interactions to algal biofuels production have not been investigated. Here, we provide a preliminary report on the frequency of co-occurrence between indole-3-acetic acid (IAA)-producing bacteria and green algae in natural and engineered ecosystems. Growth experiments with unicellular algae, Chlorella and Scenedesmus, revealed IAA concentration-dependent responses in chlorophyll content and dry weight. Importantly, discrete concentrations of IAA resulted in cell culture synchronization, suggesting that biochemical priming of cellular metabolism could vastly improve the reliability of high density cultivation. Bacterial interactions may have an important influence on algal growth and development; thus, the preservation or engineered construction of the algal-bacterial assembly could serve as a control point for achieving low input, reliable production of algal biofuels.

Cyclobacterium xiamenense sp. nov., isolated from aggregates of Chlorella autotrophica, and emended description of the genus Cyclobacterium.

A novel Gram-stain-negative, horseshoe-shaped, non-motile bacterium, designated strain KD51(T), forming colonies coloured pink by carotenoid pigments, was isolated from aggregates of the alga Chlorella autotrophica collected from the coastal sea off the city of Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison showed that strain KD51(T) was a member of the genus Cyclobacterium, forming a distinct lineage with Cyclobacterium lianum HY9(T). The 16S rRNA gene sequence similarity between strain KD51(T) and the type strains of species of the genus Cyclobacterium ranged from 92.1 % to 95.2 %. Growth occurred at 4-40 °C (optimum, 28 °C), in the presence of 3-9 % NaCl (optimum, 3-5 %) and at pH 6-10 (optimum, pH 7.5). The dominant fatty acids (>20 %) of strain KD51(T) were iso-C15 : 0 (32.2 %) and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c; 22.2 %). The DNA G+C content was 41.7 mol% and the only respiratory quinone was menaquinone-7. On the basis of phenotypic data and phylogenetic inference, strain KD51(T) represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium xiamenense sp. nov. is proposed. The type strain is KD51(T) ( = CGMCC 1.12432(T) = KCTC 32253(T)). An emended description of the genus Cyclobacterium is also proposed.

The effect of bacterial contamination on the heterotrophic cultivation of Chlorella pyrenoidosa in wastewater from the production of soybean products.

This study examined the impacts of bacteria on the algal biomass, lipid content and efficiency of wastewater treatment during the heterotrophic cultivation of Chlorella pyrenoidosa. Our results showed that soybean-processing wastewater can enhance the accumulation of lipids in algal cells and thus raise the lipid yield in the pure culture. The bacteria coexisting with algae improved the degradation of total nitrogen (TN), total phosphorus (TP), glucose and chemical oxygen demand (COD). Although the biomass productivity of algae was not significantly affected, the total algal lipid content and lipid production rate were slightly reduced when bacteria coexisted with algae. The difference in the compositions of the medium is presumed to be the main contributing factor for the variation in total lipid content in presence and absence of bacteria. The TN, TP, and COD decreased during the assimilatory process undertaken by C. pyrenoidosa, and the removal efficiency of TN by bacteria depended on the type of nitrogen species in the medium. Additionally, the apparent interaction between the bacterial and algal cultures varied with the changes in experimental conditions. Algae could compete with bacteria for the carbon and energy sources, and inhibit the growth of the bacteria in the presence of high organic matter concentration in the medium.

The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays.

Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46mugCuL(-1), while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208mugCuL(-1). However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The "axenic" cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment.

Presence of previously undescribed bacterial taxa in non-axenic Chlorella cultures.

We determined the bacterial community profile in non-axenic cultures of Chlorella (Chlorophyceae, Chlorophyta) isolated from soil. The bacterial composition at the phylum level was different from that of whole soil bacteria, but it was similar to that reported for non-axenic cultures of marine microalgae such as diatoms (Bacillariophyceae, Heterokontophyta). Expected novel bacteria, i.e. those which do not have close relatives among described species, were maintained in the cultures, and these bacteria were chiefly composed of members of the phylum Bacteroidetes. They may have been 'as-yet-uncultured' but in practice unintentionally been cultured in microalgal cultures. They could serve as good bioresources in various fields of biological and ecological studies.

Treating sewage using coimmobilized system of Chlorella pyrenoidosa and activated sludge.

Chlorella pyrenoidosa was coimmobilized with activated sludge to produce algae-bacteria beads for sewage treatment. Hydrolysis/acidogenesis pretreatment could improve the symbiotic microenvironment of coimmobilized Chlorella pyrenoidosa and activated sludge, and as a result, promote the removal of nutrients (COD(cr), inorganic nitrogen and inorganic phosphorus) in the sewage. A photo-bioreactor combining hydrolysis/acidogenesis pretreatment and coimmobilized technique was designed to treat sewage continuously. The results show that, the removal efficiencies of COD(cr), NH4(+)-N and TP reached steady state after 4-days of experiment. The removal efficiencies of COD(cr), NH4(+)-N and TP were 59.6%, 59.0% and 60.3% respectively.

Symbiotic association in Chlorella culture.

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.

Analysis of 43 kb of the Chlorella virus PBCV-1 330-kb genome: map positions 45 to 88.

Forty-three kb of DNA, located at the left end (45 to 88 kb) of the 330-kb Chlorella virus PBCV-1 genome, was sequenced and analyzed. Eighty-six open reading frames (ORFs) 65 codons or longer were identified; 47 were classified as major ORFs. These 47 major ORFs are densely packed on both strands of PBCV-1 DNA. Seventeen of these major ORFs resemble genes in the sequence databases, including three putative gene products involved in manipulating sugars (glucosamine synthetase, GDP-D-mannose dehydratase, and N-acetylglucosaminyltransferase), two transcription factors, beta-1,3-glucanase, aspartate transcarbamylase, ubiquitin carboxy terminal hydrolase, RNA guanyl transferase, an exonuclease, and a helicase. This is the first time some of these putative PBCV-1 genes have been found in a virus genome. One of the transcription factor-like genes contains a type IB self-splicing intron. Since a spliceosomal processed intron was reported previously in the PBCV-1 DNA polymerase gene, PBCV-1 is the first virus known to contain two different types of introns.