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Cyanobacteria, as photosynthetic bacteria, can generate energy and carbon sources directly from sunlight and atmospheric carbon dioxide. With the aid of genetic engineering, heterologous enzymes and/or pathways have been introduced into cyanobacteria, enabling the production of various biofuels and biochemicals. By utilizing cyanobacteria as production hosts, biofuels and biochemicals can be produced directly from carbon dioxide, and ultimately carbon dioxide fixation by cyanobacteria can help reduce atmospheric carbon dioxide concentration, alleviating global warming and air pollution. In this review, we introduce various biofuels and biochemicals produced, particularly from acetyl-CoA by cyanobacteria, and summarize research strategies that have been made to improve their production. This review will provide comprehensive information and valuable insights into strategies for enhancing cyanobacterial biofuels/chemicals production.

Cyanobacteria are promising microbial hosts for the production of diverse biofuels and biochemicals. However, compared to other model microbial hosts such as Escherichia coli and yeast, it takes a long time to genetically modify cyanobacteria. One way to efficiently engineer cyanobacteria while minimizing genetic engineering would be to develop a fast, high-throughput prototyping tool for cyanobacteria. In this study, we developed a CRISPR/Cas12a-based assay coupled with cyanobacteria cell-free systems to rapidly prototype promoter characteristics. Using this newly developed assay, we demonstrated cyanobacteria cell-free transcription for the first time and confirmed a positive correlation between the in vitro and in vivo transcription performance. Furthermore, we generated a synthetic promoter library and evaluated the characteristics of promoter subregions by using the assay. Varied promoter strength derived from random mutations were rapidly and effectively measured in a high-throughput way. We believe that this study offers an easily applicable and rapid prototyping platform to characterize promoters for cyanobacterial engineering.

Freshwater Synechocystis sp. PCC 6803 has been considered to be a platform for the production of the next generation of biofuels and is used as a model organism in various fields. Various genomics, transcriptomics, metabolomics, and proteomics studies have been performed on this strain, whereas marine Synechocystis sp. PCC 7338 has not been widely studied despite its wide distribution. This study analyzed the proteome profiles of two Synechocystis strains using a liquid chromatography–tandem mass spectrometry-based bottom-up proteomic approach. Proteomic profiling of Synechocystis sp. PCC 7338 was performed for the first time with a data-dependent acquisition method, revealing 18,779 unique peptides and 1794 protein groups. A data-independent acquisition method was carried out for the comparative quantitation of Synechocystis sp. PCC 6803 and 7338. Among 2049 quantified proteins, 185 up- and 211 down-regulated proteins were defined in Synechocystis sp. PCC 7338. Some characteristics in the proteome of Synechocystis sp. PCC 7338 were revealed, such as its adaptation to living conditions, including the down-regulation of some photosynthesis proteins, the up-regulation of kdpB, and the use of osmolyte glycine as a substrate in C1 metabolism for the regulation of carbon flow. This study will facilitate further studies on Synechocystis 7338 to define in depth the proteomic differences between it and other Synechocystis strains.

Biodiesel is a biodegradable and renewable fuel. A large amount of research has considered microbial oil production using oleaginous microorganisms, but the commercialization of microbial lipids produced in this way remains uncertain due to the high cost of feedstock or low lipid yield. Microbial lipids can be typically produced by microalgae, yeasts, and bacteria; the lipid yields of these microorganisms can be improved by using sufficient concentrations of organic carbon sources. Therefore, combining low-cost organic compounds contained in organic wastes with cultivation of oleaginous microorganisms can be a promising approach to obtain commercial viability. However, to achieve effective bioconversion of low-cost substrates to microbial lipids, the characteristics of each microorganism and each substrate should be considered simultaneously. This article discusses recent approaches to developing cost-effective microbial lipid production processes that use various oleaginous microorganisms and organic wastes.

Despite an increasing potential of red algal biomass as a feedstock, biological conversion of red algal biomass has been limited by lack of feasible microorganisms which can convert structured AHG, which is a main component of red algal carbohydrate, into a common metabolite. In the AHG uptake pathway, AHG dehydrogenase (AHGD) is known to be a key step, therefore it is important to find an efficient dehydrogenase to break down 3,6- anhydro-L-galactose (AHG) for practical use of red macroalgae biomass in biorefineries requires. In this study, we isolate a novel AHG dehydrogenase (AHGD) with high activity produced by a newly isolated bacteria strain, Raoultella ornithinolytica B6–JMP12. The stability and compatibility of the enzyme were evaluated under various conditions to achieve high enzyme production. The AHGD was partially purified using conventional protein purification techniques such as ammonium sulfate precipitation and ion exchange followed by gel filtration chromatography, 37.24 fold with a final specific activity of 5.47 U/mg of protein with 32% yield recovery. SDS-PAGE was used to determine the molecular weight of the partially purified AHGD and its molecular weight was found to be around ~34 kDa. The optimal pH and temperature for the partially purified AHGD were 7.0 and 35°C, respectively. The Km and Vmax for 3,6-anhydro-L-galactose are 0.63 mg/mL and 0.38 μM/mL/min, respectively.

The goal of this study was to investigate the influences of gradually mixotrophic culture conditions on microalgal biomass and lipid production by a consortium of indigenous microalgae and bacteria present in raw municipal wastewater. Lab-scale photobioreactors containing the consortium were operated in repeated batch mode. Initial cultivation (phase I) was performed using only the municipal wastewater, then 10% and 25% of the reactor volumes were replaced with the effluent from a sewage sludge fermentation system producing volatile fatty acids (SSFV) at the beginnings of phase II and phase III, respectively. The highest biomass productivity (117.1±2.7mg/L/d) was attained during phase II, but the lipid productivity (17.2±0.2mg/L/d) was attained during phase III. The increase in the effluent from the SSFV influenced microalgal diversity with a preference for Chlorella sp., but bacterial diversity increased significantly during phase III.

Genetic information of reproduction and growth is essential for sustainable molluscan fisheries and aquaculture management. However, there is limited knowledge regarding the reproductive activity of the commercially important Pacific abalone Haliotis discus hannai. We performed de novo transcriptome sequencing of the ganglia in sexually immature and mature female Pacific abalone to better understand the sexual maturation process and the underlying molecular mechanisms. Of the ~305 million high-quality clean reads, 76,684 transcripts were de novo-assembled with an average length of 741 bp, 28.54% of which were annotated and classified according to Gene Ontology terms. There were 256 differentially expressed genes between the immature and mature abalone. Tandem mass spectrometry analysis, as compared to the predicted-peptide database of abalone ganglia transcriptome unigenes, identified 42 neuropeptide precursors, including 29 validated by peptidomic analyses. Label-free quantification revealed differential occurrences of 18 neuropeptide families between immature and mature abalone, including achatin, FMRFamide, crustacean cardioactive peptide, and pedal peptide A and B that were significantly more frequent at the mature stage. These results represent the first significant contribution to both maturation-related transcriptomic and peptidomic resources of the Pacific abalone ganglia and provide insight into the roles of various neuropeptides in reproductive regulation in marine gastropods.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

This study demonstrates that increased NADPH production can improve biomass and ethanol production in cyanobacteria. We over-expressed the endogenous zwf gene, which encodes glucose-6-phosphate dehydrogenase of pentose phosphate pathway, in the model cyanobacterium Synechocystis sp. PCC 6803. zwf over-expression resulted in increased NADPH production, and promoted biomass production compared to the wild type in both autotrophic and mixotrophic conditions. Ethanol production pathway including NADPH-dependent alcohol dehydrogenase was also integrated with and without zwf over-expression. Excessive NADPH production by zwf over-expression could improve both biomass and ethanol production in the autotrophic conditions.

Cyanobacteria play a major role as direct producers of biofuels, such as ethanol and butanol, with the aid of genetic engineering. However, development of a new harvesting-technology is essential to achieve economic viability of biofuel production from cyanobacteria. In this study, we demonstrated the feasibility of harvesting the unicellular cyanobacterium Synechocystis sp. PCC 6803 using pre-made filamentous fungal pellets and investigated key factors affecting efficiency of harvest, including fungal strain, pellet quantity (number of pellets), initial pH, and organic carbon source. Synechocystis sp. PCC 6803 cells attached to Aspergillus oryzae pellets, indicating that this fungal pellet had a desirable harvesting effect, while Rhizopus oryzae pellets had no effect on harvesting. Increasing pellet quantity and adding organic carbon sources, such as glucose and xylose, improved the harvesting efficiency of Aspergillus oryzae pellet; efficiency was not affected by the initial pH.

This study is to investigate the characteristic features of Acanthamoeba keratitis (AK) that differentiating it from herpetic epithelial keratitis (HEK) using anterior segment optical coherence tomography (AS-OCT). Medical records of three eyes of each AK and herpetic keratitis who had AS-OCT examination were reviewed in this study. Slit-lamp biomicroscopy and AS-OCT was performed on the initial visit and on every follow-up visits in all patients. In all three AK cases, reflective bands in the corneal stroma that correspond to the area of radial keratoneuritis were observed. The depth of the reflective bands varied in each case. After AK treatment, slit-lamp biomicroscopy confirmed that radial keratoneuritis had resolved and AS-OCT confirmed that reflective bands in the corneal stroma had also disappeared in all patients. Unlike the AS-OCT results found in AK, highly reflective HEK lesions were observed only in the subepithelial area, not in the stroma. AS-OCT seems to be helpful analyzing the specific depth of the lesion which enables to distinguish AK from HEK.

Single-stage thermophilic anaerobic co-digestion (TAcoD) of waste activated sludge (WAS) and food wastewater (FWW) was used to examine effects of increase of FWW mixing ratio on the organic matter removal and populations of methanogenic archaea species. The volume percent of FWW in the feedstock (mixture of WAS and FWW) increased gradually from 0% to 100% in increments of 25%. Organic matter removal efficiency increased with FWW mixing ratio, but declined when FWW was used as the sole substrate. The highest organic matter removal and biogas production (VS removal of 77%, TCOD removal of 65.5%, methane production rate of 1422.50 mL CH4/(L d), CH4 content of 68.24%, CH4 yield of 316.11 mL CH4/g CODremoved) was achieved when FWW mixing ratio was 75%. Quantitative real-time PCR results indicated that the populations of methanogenic archaea were highest when FWW mixing ratio was 75%. Pyrosequencing analysis revealed relatively high abundances of two methanogenic genera Methanothermobacter (order Methanobacteriales) and Methanosarcina (order Methanosarcinales) throughout digestion. The changes of key methanogenic archaea correlate with organic matter removal and biogas production, and are influenced strongly by organic acid concentration.

Brown seaweed contains up to 67% of carbohydrates by dry weight and presents high potential as a polysaccharide feedstock for biofuel production. To effectively use brown seaweed as a biomass, degradation of alginate is the major challenge due to its complicated structure and low solubility in water. This study focuses on the isolation of alginate degrading bacteria, determining of the optimum fermentation conditions, as well as comparing the conventional single fermentation system with the two-phase fermentation system which is separately using alginate and mannitol extracted from Laminaria japonica. Maximum yield of organic acids production and volatile solids reduction obtained were 0.516 g/g and 79.7%, respectively, using the two-phase fermentation system in which alginate fermentation was carried out at pH 7 and mannitol fermentation at pH 8. The two-phase fermentation system increased the yield of organic acids production by 1.14 times and led to a 1.45-times reduction of VS when compared to the conventional single fermentation system at pH 8. The results show that the two-phase fermentation system improved the utilization of alginate by separating alginate from mannitol leading to enhanced alginate lyase activity.

Microalgae, Scenedesmus bijuga, was cultivated in anaerobically digested food wastewater effluent (FWE) to treat the wastewater and produce biodiesel simultaneously. Three different mixing ratios with municipal wastewater were compared for finding out proper dilution ratio in biodiesel production. Of these, 1/20 diluted FWE showed the highest biomass production (1.49 g/L). Lipid content was highest in 1/10 diluted FWE (35.06%), and the lipid productivity showed maximum value in 1/20 diluted FWE (15.59 mg/L/d). Nutrient removal was also measured in the cultivation. FAME compositions were mainly composed of C16-C18 (Over 98.94%) in S. bijuga. In addition, quality of FAMEs was evaluated by Cetane Number (CN) and Bis-allylic Position Equivalent (BAPE).

Marine biomass is a potential carbon resource for the production of bio-based energy and chemicals. Microbial lipids are competitive in producing biodiesel. In this study, Laminaria japonica ( Saccharina japonica ) was chosen as a model marine biomass producer due to its fast rate and well-established cultivation technology. A mixed culture system was established by inoculating it with sludge from an anaerobic digester, and then operated in a continuous mode for over 1000 days by feeding it a non-sterile suspension of dried L. japonica in tap water without additional nutrients. The average yield and productivity of total organic acids during the stationary phase were as high as 0.47 g/g COD and 2.45 g/L/day at pH 7 with 84.08% of acetate in total organic acids. The volatile fatty acids (VFAs) from the continuously-stirred tank reactor (CSTR) were used by oleaginous yeast ( Cryptococcus curvatus ) to produce lipids in a repeated batch system. The highest lipid content was 61%. The composition of the fatty acids was quite similar to that of vegetable oils. This system has potential application to biodiesel production.

Volatile fatty acid (VFA) production, utilization of soluble organic compounds, and associated microbial consortia were investigated after different pretreatments (untreated, alkaline, and thermal-alkaline) using cyanobacterial biomass as a substrate. Compared to the untreated control, soluble carbohydrate concentrations were almost the same after alkaline and thermal-alkaline pretreatments, but soluble protein concentration was 1.58 times higher after alkaline pretreatment and 1.81 times higher after thermal-alkaline pretreatment. However, the highest degree of acidification was obtained after alkaline pretreatment (55.36 ± 3.00%). Microbial communities in the untreated control differed only slightly from those after thermal-alkaline pretreatment, but were clearly distinct from those after alkaline pretreatment. After alkaline pretreatment, protein-utilizing bacteria became relatively predominant. These results revealed the relationships between efficiency of VFA production and the shift in microbial community.

The aims of this study were to quantify how pretreatment affects production of volatile fatty acids (VFAs) from cyanobacterial biomass and production of subsequent microbial lipid by an oleaginous microorganism that uses the VFAs as carbon sources. The highest biomass solubilization was obtained using thermal-alkaline (th-alkaline) pretreatment (33.1%), followed by alkaline pretreatment (29.1%), and thermal pretreatment (7.2%), but the highest VFA yield was obtained using alkaline pretreatment (0.54±0.02g/gVS), followed by the untreated condition (0.47±0.03g/gVS), and th-alkaline pretreatment (0.44±0.02g/gVS). Although VFA yield was higher using alkaline pretreatment condition than in the untreated condition, the difference was not great. However, lipid productivity by Cryptococcus curvatus after the alkaline pretreatment condition was 2.0-fold higher than that under the untreated condition. This study confirmed the feasibility of using biologically produced VFAs from cyanobacterial biomass for microbial lipid production by the oleaginous microorganism.

As aquatic biomass which is called "the third generation biomass", Laminaria japonica (also known as Saccharina japonica) consists of mannitol and alginate which are the main polysaccharides of algal carbohydrates. In this study, oleaginous yeast (Cryptococcus curvatus) was used to produce lipid from carbon sources derived from Laminaria japonica. Volatile fatty acids (VFAs) were produced by fermentation of alginate extracted from L. japonica. Thereafter, mannitol was mixed with VFAs to culture the oleaginous yeast. The highest lipid content was 48.30%. The composition of the fatty acids was similar to vegetable oils. This is the first confirmation of the feasibility of using macroalgae as a carbon source for biodiesel production.

A lab-scale sequential sludge digestion process which consists of a mesophilic anaerobic digester (MAD) and a thermophilic aerobic digester (TAD) was developed. Thermal, thermal–alkaline and long-term alkaline pre-treatments were applied to the feed sludge to examine their effects on sludge removal and methane production. Especially after thermal–alkaline pre-treatment, high COD removal was maintained; methane production rate was also drastically increased by improving the hydrolysis step of sludge degradation. Polymerase chain reaction–denaturing gel gradient electrophoresis indicated that bacterial communities were represented by three phyla (Firmicutes, Proteobacteria, Actinobacteria) and that Clostridium straminisolvens was the major bacterial species in MAD. Quantitative real-time PCR results indicated that Methanosaeta concilli was the major archaeal species in MAD, and that Ureibacillus sp. was the most abundant bacterial species in TAD.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that 'intrinsic component of membrane' was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in 'secondary metabolites biosynthesis, transport and catabolism' and 'signal transduction mechanisms' in the KOG clusters and 'processing in endoplasmic reticulum', and 'ABC transporters' in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

To evaluate the applicability of single-stage thermophilic aerobic digestion (TAD) process treating high-strength food wastewater (FWW), TAD process was operated at four organic loading rates (OLRs) from 9.2 to 37.2 kg COD/m 3 d. The effects of OLRs on microbial community changes were also examined. The highest volumetric removal rate (13.3 kg COD/m 3 d) and the highest thermo-stable protease activity (0.95 unit/mL) were detected at OLR = 18.6 kg COD/m 3 d. Denaturing gradient gel electrophoresis (DGGE) profiles and quantitative PCR (qPCR) results showed significant microbial community shifts in response to changes in OLR. In particular, DGGE and phylogenetic analysis demonstrate that the presence of Bacillus sp. (phylum of Firmicutes) was strongly correlated with efficient removal of organic particulates from high-strength food wastewater.

An effective two-stage sewage sludge digestion process, consisting of thermophilic aerobic digestion (TAD) followed by mesophilic anaerobic digestion (MAD), was developed for efficient sludge reduction and methane production. Using TAD as a biological pretreatment, the total volatile suspended solid reduction (VSSR) and methane production rate (MPR) in the MAD reactor were significantly improved. According to denaturing gradient gel electrophoresis (DGGE) analysis, the results indicated that the dominant bacteria species such as Ureibacillus thermophiles and Bacterium thermus in TAD were major routes for enhancing soluble organic matter. TAD pretreatment using a relatively short SRT of 1 day showed highly increased soluble organic products and positively affected an increment of bacteria populations which performed interrelated microbial metabolisms with methanogenic species in the MAD; consequently, a quantitative real-time PCR indicated greatly increased Methanosarcinales (acetate-utilizing methanogens) in the MAD, resulting in enhanced methane production.

To investigate the microorganisms in culture-proven endophthalmitis and their susceptibilities to antimicrobial agents commonly used in South Korea.Medical records of consecutive patients with culture-proven endophthalmitis at eight institutions between 1 January 2004 and 31 July 31 2010 were reviewed. Four categories of endophthalmitis were studied: postoperative, posttraumatic, endogenous, and unspecified. Outcome measures were culture-proven infectious organisms, antimicrobial susceptibilities, and final visual acuity in the patients.A total of 93 microorganisms were identified from 103 patients during the study period. The positive culture rate was 59.2 % (103/174). The most common organisms identified were Enterococcus faecalis (in 20.8 % of patients, 20/96), Staphylococcus epidermidis (18.8 %, 18/96), other coagulase-negative staphylococci (10.4 %, 10/96), Pseudomonas aeruginosa (6.3 %, 6/96), and Klebsiella pneumoniae (6.3 %, 6/96). Two cases of Enterococcus faecium (2.1 %) were recognized. Overall, 70 of 96 (73.0 %) isolates were Gram-positive bacteria, 22 (23.0 %) were Gram-negative bacteria, and 4 (4.2 %) were fungi. The most common organisms resulting in reduced light perception were E. faecalis and K. pneumoniae.The emergence of E. faecalis in endophthalmitis is mainly caused by the high incidence of E. faecalis in postoperative endophthalmitis. This increase also impacts the final visual acuity of the patients.

In this study a new bacterium capable of growing on brown seaweed Laminaria japonica, Enterobacter sp. JMP3 was isolated from the gut of turban shell, Batillus cornutus. In anaerobic condition, it produced high yields of ethanol (1.15 mol-EtOH mol-mannitol−1) as well as organic acids from mannitol, the major carbohydrate component of L. japonica. Based on carbon distribution and metabolic flux analysis, it was revealed that mannitol was more favorable than glucose for ethanol production due to their different redox states. This indicates that L. japonica is one of the promising feedstock for bioethanol production. Additionally, the mannitol dehydrogenation pathway in Enterobacter sp. JMP3 was examined and verified. Finally, an attempt was made to explore the possibility of controlling ethanol production by altering the redox potential via addition of external NADH in mannitol fermentation.

Over a 3 year period, interactions between nitrifying bacterial communities and the operational parameters of a full-scale wastewater treatment plant were analyzed to assess their impact on nitrification performance. Throughout the study period, nitrification fluctuated while Nitrosomonas europaea and Nitrosomonas nitrosa, the two major ammonia oxidizing bacteria (AOB) communities, showed resistance to changes in operational and environmental conditions. Nitrobacter populations mostly exceeded those of Nitrospira within nitrite oxidizing bacteria (NOB). Meanwhile, principal component analysis (PCA) results revealed that a close association between Nitrobacter and nitrite concentration as well as a direct correlation between the quantity of AOB and influent SCN- concentration. The serial shifts of data points over time showed that the nitrification of a full-scale treatment plant has been gradually suppressed by the influence of influent COD and phenol concentrations.

The two-stage culture system consisting of green vegetative growth and reddish inductive production stages has been widely accepted for the production of astaxanthin using Haematococcus pluvialis. However, little has been known about the appropriate cellular phase of H. pluvialis for transferring into the astaxanthin inductive conditions. In this study, we determined the optimal growth phase of H. pluvialis for transferring into the second production stage. Astaxanthin productivities were correlated with growth phases, as senescent green phases could increase more than 10-fold greater than juvenile green phases. Our results clearly demonstrated the appropriateness of the senescent vegetable cells for transferring into the production stage, due to the increased capacity to accumulate astaxanthin.

To improve the efficiency of total nitrogen (TN) removal, solid retention time (SRT) and internal recycling ratio controls were selected as operating parameters in a full-scale activated sludge process treating high strength industrial wastewater. Increased biomass concentration via SRT control enhanced TN removal. Also, decreasing the internal recycling ratio restored the nitrification process, which had been inhibited by phenol shock loading. Therefore, physiological alteration of the bacterial populations by application of specific operational strategies may stabilize the activated sludge process. Additionally, two dominant ammonia oxidizing bacteria (AOB) populations, Nitrosomonas europaea and Nitrosomonas nitrosa, were observed in all samples with no change in the community composition of AOB. In a nitrification tank, it was observed that the Nitrobacter populations consistently exceeded those of the Nitrospira within the nitrite oxidizing bacteria (NOB) community. Through using quantitative real-time PCR (qPCR), nirS, the nitrite reducing functional gene, was observed to predominate in the activated sludge of an anoxic tank, whereas there was the least amount of the narG gene, the nitrate reducing functional gene.

An internally radiating photobioreactor was applied for the production of astaxanthin using the unicellular green alga Haematococcus pluvialis. The cellular morphology of H. pluvialis was significantly affected by the intensity of irradiance of the photobioreactor. Small green cells were widespread under lower light intensity, whereas big reddish cells were predominant under high light intensity. For these reasons, growth reflected by cell number or dry weight varied markedly with light conditions. Even under internal illumination of the photobioreactor, light penetration was significantly decreased as algal cells grew. Therefore, we employed a multistage process by gradually increasing the internal illuminations for astaxanthin production. Our results revealed that a multistage process might be essential to the successful operation of a photobioreactor for astaxnthin production using H. pluvialis.

This study was conducted to isolate microorganisms from the gut of the marine turban shell, Batillus cornutus, which inhabits the mainland of South Korea and primarily feeds on brown algae. We were interested in isolating such gut bacteria by considering their potential to produce the polysaccharidases required for digestion of brown seaweeds and isolated three different bacteria from the gut of Batillus cornutus. The isolated bacteria were identified as Bacillus sp. JMP-A, Bacillus sp. JMP-B and Staphylococcus sp. JMP-C. The organisms were evaluated for their ability to produce polysaccharidases such as cellulase, alginate lyase, laminarinase and kelp-lyase. Bacillus sp. JMP-A and Bacillus sp. JMP-B showed a clear zone of CMC hydrolysis with a radius 1.10 (±0.057) and 3.88 cm (±0.088), respectively, whereas Staphylococcus sp. JMP-C showed no zone of CMC hydrolysis. SEM analysis confirmed that the ability of the bacterial isolates to degrade kelp differs and is correlated with kelp-lyase production. The cell free extract of the Bacillus sp. JMP-A isolate showed the highest activities of CM-cellulase, α-cellulase, laminarinase and kelp-lyase, which were 22.76, 27.10, 66.59 and 64.36 U/mg, respectively. Meanwhile, the amount of sugars released was higher during the saccharification of kelp by dialyzed intracellular enzymes of the bacterial isolates than when dialyzed extracellular enzyme was used. Experimental results of dialyzed enzymatic saccharification of the kelp demonstrated that use of partially purified enzymes was effective for glucose production.

A novel biomass, dried leaves of Pinus densiflora, showed very good performance in removing mutagenic Cr(VI) from aqueous solution in this study. Analytical results for different chromium species in aqueous and solid phases indicated that the mechanism of Cr(VI) removal by the biomass was ‘adsorption-coupled reduction’. Among various parameters of batch operation, pH, temperature and contact time significantly affected Cr(VI) removal and total Cr removal individually. As a result, optimal condition for the Cr(VI) removal was not equal to that for the total Cr removal. To examine the individual effects of these parameters on the Cr(VI) and total Cr removals statistically, therefore Box–Behnken model for experiment design and second-order polynomial model for fitting experimental data were used in this study. The removal efficiency of Cr(VI) increased with a decrease in pH or with increases of temperature and contact time until equilibrium had been attained. Meanwhile, an optimum pH existed for the total Cr removal efficiency, but increased with the increases of temperature and contact time. With 60 h of contact time, 100% of Cr(VI) removal and > 95% of total Cr removal could be obtained at pH 4 and 40 °C.

Changes in process performance and the nitrifying bacterial community associated with an increase of thiocyanate (SCN-) loading were investigated in a pre-denitrification process treating industrial wastewater. The increased SCN- loading led to the concentration of total nitrogen (TN) in the final effluent, but increasing the internal recycling ratio as an operation parameter from 2 to 5 resulted in a 21% increase in TN removal efficiency. In the aerobic reactor, we found that the Nitrosomonas europaea lineage was the predominant ammonia oxidizing bacteria (AOB) and the percentages of the AOB population within the total bacteria increased from about 4.0% to 17% with increased SCN- concentration. The increase of nitrite loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrospira over Nitrobacter. Meanwhile, a Thiobacillus thioparus was suggested to be the main microorganism responsible for the SCN- biodegradation observed in the system.

Real-time quantitative PCR (qPCR) was applied to determine plasmid stability in recombinant Clostridium tyrobutyricum harboring pJIR418 shuttle vector (ATCC 77387). For the detection of the plasmid and the host chromosomal DNA, two primer sets were designed specifically for the plasmid chloramphenicol-resistant gene (cml) and for the 16S rRNA gene (16S), respectively. The plasmid copy number can be determined as the copy ratio of cml to 16S. The qPCR assay was able to evaluate the plasmid stability with good reproducibility and high sensitivity in non-selective conditions during a continuous fermentation.

Two-stage anaerobic digestion process has been frequently applied to the sequential production of hydrogen and methane from various organic substrates/wastes. In this study, a cost-effective byproduct of food industry, molasses, was used as a sole carbon source for the two-stage biogas-producing process. The two-stage process consisted of two reactor parts named as the first-stage hydrogenic reactor (HR) operated at pH 5.5 and 35°C and the second-stage methanogenic reactor (MR) at pH 7.0 and 35°C. Microbial community analysis revealed that Clostridium butyricum was the major hydrogen-producing bacteria and methanogens consisted of hydrotrophic bacteria like Methanobacterium beijingense and acetotrophic bacteria like Methanothrix soehngenii . In the first-stage process, hydrogen could be efficiently produced from diluted molasses with the highest production rate of 2.8 (±0.22) L-H 2 /L-reactor/d at the optimum HRT of 6 h. In the second-stage process, methane could be also produced from residual sugars and VFAs with a production rate of 1.48 (±0.09) L-CH 4 /L-reactor/d at the optimum HRT of 6 d, at which overall COD removal efficiency of the two-stage process was determined to be 79.8%. Finally, economic assessment supported that cost-effective molasses was a potent carbon source for the sequential production of hydrogen and methane by two-stage anaerobic digestion process.

Angelica gigas root cultures were elicited with various elicitors, including yeast extract, chitin, methyl jasmonate, salicylic acid, and copper, with the aim of increasing the production of decursin and decursinol angelate. The treatment of A. gigas root cultures with a combination of yeast extract (2 g l−1) and copper ion (0.5 mM) at the late exponential growth phase increased decursinol angelate accumulation up to 6.86 mg l−1. The best elicitor preparation selected through in vitro experiments was also applied to roots of A. gigas whole plants grown in the field in order to investigate the potential of elicitation as a novel cultivation practice for producing medicinal herbs of improved quality. Biweekly treatments with the elicitor at 70 mg g l−1 FW roots for 10 weeks before the annual harvest resulted in an increment in both plant yields and specific productivity of decursins by 1.5- and 1.7-fold, respectively. This result implies that in vitro screening of elicitors with the ultimate aim of in planta elicitation of whole plants could be effective in terms of time and expense. The elicitation technique reported here demonstrates it potential as a strategy for improving growth and active compounds productivity of medicinal plants through short-term and pre-harvest treatment of the elicitor preparation.

The H2-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum. A 2327-bp DNA region containing the ORF and the putative promoter region was amplified and subcloned into a pJIR418 shuttle vector. The gene transfer of the recombinant plasmid into C. tyrobutyricum JM1 was performed by a modified electrotransformation method. Homologous overexpression of the [FeFe]-hydrogenase gene resulted in a 1.7-fold and 1.5-fold increase in hydrogenase activity and hydrogen yield concomitant with the shift of metabolic pathway.

Three different bacteria capable of degrading phenanthrene were isolated from sludge of a pulp wastewater treatment plant and identified as Acinetobacter baumannii , Klebsiella oxytoca , and Stenotrophomonas maltophilia . Phenanthrene degradation efficiencies by different combinations (consortia) of these bacteria were investigated and their population dynamics during phenanthrene degradation were monitored using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP). When a single microorganism was used, phenanthrene degradation efficiency was very low (48.0, 11.0, and 9.0% for A. baumannii , K. oxytoca , and S. maltophilia respectively, after 360 h cultivation). All consortia that included S. maltophilia degraded ∼80.0% of phenanthrene and reduced lag time to 48 h compared to the 168 h of pure A. baumannii culture. CE-SSCP analysis showed that S. maltophilia was the predominant species during phenanthrene degradation in the mixed culture. The results indicate that mixed cultures of microorganisms may effectively degrade target chemicals, even if the microorganisms show low degradation activity in pure culture.

The effects of pH on hydrogen fermentation of glucose by newly isolated H(2)-producing bacterium Clostridium tyrobutyricum JM1 were investigated in batch cultivations. The changes of carbon material and energy balances by pH conditions provided useful information for understanding and interpreting the regulatory system of the microorganism, and for optimization of a desired product, in this case, molecular hydrogen. The most probable metabolic pathways of C. tyrobutyricum JM1 were determined through an accurate analysis of stoichiometry and the consistency of the experimental data, checked by high carbon recovery. The carbon material and energy balances were adequately applied to estimate the carbon-flow distribution. They suggested that pH 6.3 was appropriate to maximize hydrogen production with a high concentration of butyrate and balanced activities of NADH.

A fermentative hydrogen-producing bacterium was isolated from a food waste treatment process. The biological hydrogen production rate by the pure isolate (designated as Clostridium tyrobutyricum JM1) was dependent on various nutritional and environmental conditions. In this study, to enhance hydrogen production rate, the individual and mutual effects of three key process variables such as glucose concentration, pH and temperature were investigated through response surface methodology (RSM) in a batch system. A Box–Behnken design was employed to determine the effect of the three independent variables on the hydrogen production rate and to find the optimum condition of each variable for improved hydrogen production. Experimental results showed that a maximum hydrogen production rate of 5089 ml H 2 (g dry cell h) −1 was obtained under the condition of glucose concentration of 102.08 mM, temperature 35 °C and pH 6.5, and all three factors had significant influences on the specific hydrogen production rate. The RSM with the Box–Behnken design was a useful tool for achieving the high rate of hydrogen production by C. tyrobutyricum JM1.

A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2 l H2 l−1 d−1 at 2 h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223 ml (g-hexose)−1 with an influent glucose concentration of 5 g l−1. Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production.

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box–Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H 2 (g dry cell h) −1 was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 °C and pH 6.13. The experimental results showed that the RSM with the Box–Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes .

A simple and efficient protocol was developed for culturing Cu-tolerant and Cu-accumulating plants via pre-adaptation to Cu during plant tissue culture. We induced multiple shoots from begonia (Begonia evansiana Andr.) leaf explants on MS medium supplemented with naphtaieneacetic acid and benzyladenine. After 3 months, small plantlets were transferred to MS medium supplemented with for pre-adaptation to Cu and cultured for 5 months. Then, these plantlets were individually planted in pots containing artificial soil. An additional 500 mg of Cu dissolved in 1/4 strength MS solution was applied to each pot during irrigation over the course of 2 months. We planted pre-adapted and control begonias in soil from the II-Kwang Mine, an abandoned Cu mine in Pusan, Korea, to examine their ability to tolerate and accumulate Cu for phytore-mediation. Pre-adapted begonias accumulated Cu/g dry root tissue over the course of 45 days. On the other hand, non-Cu-adapted controls accumulated only Cu/g dry root tissue. To enhance Cu extraction, chelating agents, ethylenediamine tetraacetic acid (EDTA)-dipotassiun and pyridine-2,6-dicarboxylic acid (PDA), were applied. While the chelating agents did not enhance accumulation of Cu in the roots of control begonias, EDTA application increased the level of Cu in the roots of pre-adapted begonias twofold (to Cu/g dry root tissue). Because pre-adapted begonias accumulated a large amount of Cu, mainly in their roots, they could be used for phytostabilization of Cu-contaminated soils. In addition, as a flowering plant, begonias can be used to create aesthetically pleasing remediation sites.

The dead biomass of the brown seaweed, Ecklonia sp., is capable of reducing toxic Cr(VI) into less toxic or nontoxic Cr(III). However, little is known about the mechanism of Cr(VI) reduction by the biomass. The objective of this work was to develop a kinetic model for Cr(VI) biosorption, for supporting our mechanism. The reduction rate of Cr(VI) increased with increasing total chromate concentration, [Cr(VI)], and equivalent concentration of organic compounds, [OCs], and decreasing solution pH. It was found that the reduction rate of Cr(VI) was proportional to [Cr(VI)] and [OCs], suggesting the simple kinetic equation -d[Cr(VI)]/dt=k[Cr(VI)][OCs]. When considering the consumption of organic compounds due to the oxidation by Cr(VI), an average rate coefficient of 9.33 (+/-0.65)microM(-1)h(-1) was determined, at pH 2. Although the function of the pH could not be expressed in a mechanistic manner, an empirical model able to describe the pH dependence was obtained. It is expected that the developed rate equation could likely be used for design and performance predictions of biosorption processes for treating chromate wastewaters.

In this study, the removal of Cr(VI) was examined in a binary aqueous system containing Ni(II), and the competitive interaction between them was successfully modeled. The removal rate of Cr(VI) was unaffected by the presence of Ni(II). However, in an equilibrium state, the uptake of total Cr or Ni(II) was decreased in the presence of the other metal. The mono- and multi-component Langmuir adsorption models clearly represented the uptake behavior of these metals.

Brown seaweed Ecklonia biomass was used for the treatment of electroplating wastewater that contains chromium and zinc ions. Batch experiments showed that Cr(VI) was removed from the wastewater through reduction to Cr(III) by contact with the biomass, whereas Cr(III) and Zn(II) were removed through adsorption to the binding sites of the biomass. Among various parameters, the solution pH most significantly affected the biosorptive capacity of the biomass. As the solution pH increased, the removal efficiency of Cr(VI) decreased, whereas that of Cr(III) and Zn(II) increased, for pH

Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH(4+) 8.00 mM, PO(4)(3-) 1.11 mM, and SO(4)(2-) 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h(-1). Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H(2) for a 10-mL culture of alga with density of 6 x 10(6) cells mL(-1) for 96 h under conditions of NH(4)(+) 9.20 mM, PO(4)(3-) 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency.

The removal of hexavalent chromium from aqueous solution was carried out in batch experiments using dead biomass of four fungal strains – Aspergillus niger , Rhizopus oryzae , Saccharomyces cerevisiae and Penicillium chrysogenum . All of these dead fungal biomass completely removed Cr(VI) from aqueous solutions, that of R . oryzae being the most effective. Cr(VI) was removed from aqueous solutions by the reduction to Cr(III) when it contacted with the biomass. The removal rate of Cr(VI) increased with a decrease in pH or with increases of Cr(VI) and biomass concentrations. In particular, the removal rate of Cr(VI) was proportional to total chromate concentration [Cr(VI)], and equivalent concentration of organic compounds [OC], suggesting a simple rate equation in a form of d[Cr(VI)]/d t = − k [Cr(VI)][OC]. This model fitted well with the experimental data obtained at pH 2, supporting the mechanism that Cr(VI) is removed via a redox reaction. From the practical view point, the abundant and inexpensive dead fungal biomass could be used for the conversion of toxic Cr(VI) into less toxic or nontoxic Cr(III).

When synthetic wastewater containing Cr(VI) was placed in contact with the dead fungal biomass of Aspergillus niger, the Cr(VI) was completely removed from aqueous solution, whereas Cr(III), which was not initially present, appeared in aqueous solution. Desorption and X-ray photoelectron spectroscopy (XPS) studies showed that most of the Cr bound on the biomass was in trivalent form. These results indicated that the main mechanism of Cr(VI) removal was a redox reaction between Cr(VI) and the dead fungal biomass, which is quite different from previously reported mechanisms. The influences of contact time, pH, Cr(VI) concentration, biomass concentration and temperature on Cr(VI) removal were also evaluated. The Cr(VI) removal rate increased with a decrease in pH and with increases in Cr(VI) concentration, biomass concentration and temperature. Although removal kinetics was dependent on the experimental conditions, Cr(VI) was completely removed in the aqueous solution. In conclusion, a new mechanism of Cr(VI) removal by the dead fungal biomass has been proposed. From a practical viewpoint, this abundant and inexpensive dead fungal biomass has potential application in the conversion of toxic Cr(VI) into less toxic or nontoxic Cr(III).