Design of static mixers for inclined tubular photobioreactors

Static mixers, which improve gas-liquid mass transfer inside tubular photobioreactors and move the cells between the upper and lower parts of the tubes were designed. Each static mixer was equipped with an opening (v-cut slit) at the top (for gas dispersion) and an orifice at the lower part (for liquid circulation). When the static mixers were installed in the riser tube of an inclined tubular photobioreactor, vertical movement of the liquid was induced so that cells were moved between the surface and bottom part of the photobioreactor. The mass transfer rates in the tubular photobioreactor without static mixers decreased sharply when they were scaled up by increasing the tube diameters. However, by installation of static mixers, the mass transfer rates in 12.5-cm diameter tubes were almost as high as those of 3.8-cm tubes without static mixers. The effectiveness of the static mixers in improving the mass transfer characteristics of the tubular photobioreactors was higher in large than small diameter tubes. The ratio of the diameters of the static mixers to the diameter of the tube, the areas of the v-cut slits and the orifice were the important design parameters that affected the mass transfer characteristics. The gas hold up and and k_La were higher when the areas of the slits (v-cut) and the orifice were reduced.     

Light/dark cyclic movement of algal culture (<Emphasis Type="Italic">Synechocystis aquatilis</Emphasis>) in outdoor inclined tubular photobioreactor equipped with static mixers for efficient production of biomass

Synechocystis aquatilis SI-2 was grown outdoors in a 12.5cm diam. tubular photobioreactor equipped with static mixers. The static mixers ensured that cells were efficiently circulated between the upper (illuminated) and lower (dark) sections of the tubes. The biomass productivity varied from 22 to 45g m^–2d^–1, with an average of 35g m^–2d^–1, etc which corresponded to average CO₂ fixation rate of about 57 g CO₂ m^–2 d^–1. The static mixers not only helped in improving the biomass productivities but also have a high potential to lower the photoinhibitory effect of light during the outdoor cultures of algae. Revisions requested 27 July 2004; Revisions received 12 November 2004     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

Growth kinetics of microalgae in microfluidic static droplet arrays

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter‐scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell‐size distribution of the microalgae was correlated with different stages of the growth. The single‐cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day^?1 for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk‐scale experiment was 1.12 day^?1. It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell‐size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells. Biotechnol. Bioeng. 2012; 109: 2987–2996. © 2012 Wiley Periodicals, Inc.     

Study of hydrodynamic characteristics in tubular photobioreactors

In this work, the hydrodynamic characteristics in tubular photobioreactors with a series of helical static mixers built-in were numerically investigated using computational fluid dynamics (CFD). The influences of height and screw pitch of the helical static mixer and fluid inlet velocity on the cell trajectories, swirl numbers and energy consumption were examined. In order to verify the actual results for cultivation of microalgae, cultivation experiments of freshwater Chlorella sp. were carried out in photobioreactor with and without helical static mixer built-in at the same time. It was shown that with built-in helical static mixer, the mixing of fluid could be intensified, and the light/dark cycle could also be achieved which is of benefit for the growth of microalgae. The biomass productivity of Chlorella sp. in tubular photobioreactor with helical static mixer built-in was 37.26 % higher than that in the photobioreactor without helical static mixer.     

An <Emphasis Type="Italic">in silico</Emphasis> analysis of the mitochondrial protein import apparatus of plants

Background  

An in silico analysis of the mitochondrial protein import apparatus from a variety of species; including Chlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens, Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine if components differed within and between plant and non-plant species.     

Improvement of mass transfer characteristics and productivities of inclined tubular photobioreactors by installation of internal static mixers

The feasibility of improving mass transfer characteristics of inclined tubular photobioreactors by installation of static mixers was investigated. The mass transfer characteristics of the tubular photobioreactor varied depending on the type (shape) and the number of static mixers. The volumetric oxygen transfer coefficient ( k(L)a) and gas hold up of the photobioreactor with internal static mixers were significantly higher than those of the photobioreactor without static mixers. The k(L)a and gas hold up increased with the number of static mixers but the mixing time became longer due to restricted liquid flow through the static mixers. By installing the static mixers, the liquid flow changed from plug flow to turbulent mixing so that cells were moved between the surface and bottom of the photobioreactor. In outdoor culture of Chlorella sorokiniana, the photobioreactor with static mixers gave higher biomass productivities irrespective of the standing biomass concentration and solar radiation. The effectiveness of the static mixers (average percentage increase in the productivities of the photobioreactor with static mixers over the productivities obtained without static mixers) was higher at higher standing biomass concentrations and on cloudy days (solar radiation below 6 MJ m(-2) day(-1)).     

A loop‐to‐base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159

The first step in microRNA (miRNA) biogenesis usually involves cleavage at the base of its fold‐back precursor. Here, we describe a non‐canonical processing mechanism for miRNAs miR319 and miR159 in Arabidopsis thaliana. We found that their biogenesis begins with the cleavage of the loop, instead of the usual cut at the base of the stem–loop structure. DICER‐LIKE 1 (DCL1) proceeds then with three additional cuts until the mature miRNA is released. We further show that the conserved upper stem of the miR319 precursor is essential to organize its biogenesis, whereas sequences below the miRNA/miRNA^* region are dispensable. In addition, the bulges present in the fold‐back structure reduce the accumulation of small RNAs other than the miRNA. The biogenesis of miR319 is conserved in the moss Physcomitrella patens, showing that this processing mechanism is ancient. These results provide new insights into the plasticity of small‐RNA pathways.     

Energy from algae using microbial fuel cells

Bioelectricity production from a phytoplankton, Chlorella vulgaris, and a macrophyte, Ulva lactuca was examined in single chamber microbial fuel cells (MFCs). MFCs were fed with the two algae (as powders), obtaining differences in energy recovery, degradation efficiency, and power densities. C. vulgaris produced more energy generation per substrate mass (2.5 kWh/kg), but U. lactuca was degraded more completely over a batch cycle (73 ± 1% COD). Maximum power densities obtained using either single cycle or multiple cycle methods were 0.98 W/m² (277 W/m³) using C. vulgaris, and 0.76 W/m² (215 W/m³) using U. lactuca. Polarization curves obtained using a common method of linear sweep voltammetry (LSV) overestimated maximum power densities at a scan rate of 1 mV/s. At 0.1 mV/s, however, the LSV polarization data was in better agreement with single‐ and multiple‐cycle polarization curves. The fingerprints of microbial communities developed in reactors had only 11% similarity to inocula and clustered according to the type of bioprocess used. These results demonstrate that algae can in principle, be used as a renewable source of electricity production in MFCs. Biotechnol. Bioeng. 2009;103: 1068–1076. © 2009 Wiley Periodicals, Inc.     

Promoter subfragments of the sugar beet V-type H+-ATPase subunit c isoform drive the expression of transgenes in the moss Physcomitrella patens

Based on the relative ease of performing targeted nuclear gene knockout, the moss Physcomitrella patens has recently been developed as a model system for plant functional genomics. To address the need for new promoters that could drive expression of transgenes in this moss, we tested two fragments of the promoter region of the gene for the sugar beet ( Beta vulgaris) V-type H ⁺-ATPase subunit isoform c. Four gene knockout constructs were tested in which the neomycin phosphotransferase II selection marker gene was put under the control of two distinct V-type H ⁺-ATPase promoter fragments, the NOS promoter, or the CaMV 35S promoter. In each case the selection cassettes were flanked by moss FtsZ1 cDNA sequences to facilitate chromosomal targeting. From a total of more than 440 transformed plants, the number of plants generated per construct was monitored and found to be in the range of 5 to 11 stable transgenics per transformation. Both V-type H ⁺-ATPase promoter fragments lead to NPTII expression levels that were sufficiently high to generate large numbers of stable transgenic plants. The numbers of plants obtained with the two V-type H ⁺-ATPase promoter fragments were comparable to those with constructs containing the standard NOS and 35S promoters. We propose that the higher plant V-type H ⁺-ATPase promoter can be used for the expression of transgenes in the bryophyte P. patens.     

Hydroxyproline O‐arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens

Hydroxyproline O‐arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant‐specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall‐associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co‐opted to function in diverse species‐specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell‐wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall‐associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.     

Modeling and kinetic parameter estimation of alcohol dehydrogenase‐catalyzed hexanol oxidation in a microreactor

A mathematical model for hexanol oxidation catalyzed by NAD⁺‐dependent alcohol dehydrogenase from baker's yeast in a microreactor was developed and compared with the model when the reaction takes place in a macroscopic reactor. The enzyme kinetics was modeled as a pseudo‐homogeneous process with the double substrate Michaelis–Menten rate expression. In comparison with the kinetic parameters estimated in the cuvette, a 30‐fold higher maximum reaction rate and a relatively small change in the saturation constants are observed for the kinetic parameters estimated in the continuously operated tubular microreactor (V_m1=197.275 U/mg, K_m^hexanol=9.420 mmol/L, and K_m1^NAD+=0.187 mmol/L). Kinetic measurements performed in the microreactor, estimated from the initial reaction rate experiments at the residence time of 36 s, showed no product inhibition, which could be explained by hydrodynamic effects and the continuous removal of inhibiting products. The Fourier amplitude sensitivity test method was applied for global kinetic parameter analysis, which shows a significant increase in the sensitivity of K_m1^NAD+ in the microreactor. Independent experiments performed in the microreactor were used to validate and to verify the developed mathematical model.     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Effect of nanoparticle on cellular growth and lipid production in Chlorella vulgaris culture

Magnetic cobalt ferrite/silica nanoparticles (MSNs) and methyl functionalized MSNs (methyl‐MSNs) were used to enhance lipid production in Chlorella vulgaris culture through enhancement of gas‐water mass transfer and increased dissolved concentration of CO₂. Methyl‐MSNs enhanced CO₂–water mass transfer rate better than MSNs, and 0.3 wt% methyl‐MSNs are more effective than 0.1 wt% MSNs. In the cultivation experiment, 0.3 wt% methyl‐MSNs yielded the highest dry cell weight and subsequently, the highest mass transfer rate. However, enhancement of mass transfer rate did not increase lipid content. The volumetric lipid productivity in C. vulgaris culture depends not only on intracellular lipid content but also on the cell mass concentration. Consequently, 0.1 wt% methyl‐MSNs yielded the highest volumetric lipid productivity in C. vulgaris cultivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:929–933, 2018     

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.