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We introduce a non-lithographical and vacuum-free method to pattern silicon. The method combines inkjet printing and metal assisted chemical etching (MaCE); we call this method "INKMAC". A commercial silver ink is printed on top of a silicon surface to create the catalytic patterns for MaCE. The MaCE process leaves behind a set of silicon nanowires in the shape of the inkjet printed micrometer scale pattern. We further show how a potassium hydroxide (KOH) wet etching process can be used to rapidly etch away the nanowires, producing fully opened cavities and channels in the shape of the original printed pattern. We show how the printed lines (width 50⁻100 µm) can be etched into functional silicon microfluidic channels with different depths (10⁻40 µm) with aspect ratios close to one. We also used individual droplets (minimum diameter 30 µm) to produce cavities with a depth of 60 µm and an aspect ratio of two. Further, we discuss using the structured silicon substrate as a template for polymer replication to produce superhydrophobic surfaces.

Aims: The microbiota at industrial full-scale composting plants has earlier been fragmentarily studied with molecular methods. Here, fungal communities from different stages of a full-scale and a pilot-scale composting reactors were studied before and after wood ash amendment., Methods and Result: The portion of fungal biomass, determined using phospholipid fatty acid analysis, varied between 6.3% and 38.5% in different composting phases. The fungal internal transcribed spacer (ITS) area was cloned and sequenced from 19 samples representing different stages of the composting processes. Altogether 2986 sequenced clones were grouped into 166 phylotypes from which 35% had a close match in the sequence databases. The fungal communities of the samples were related with the measured environmental variables in order to identify phylotypes typical of certain composting conditions. The fungal phylotypes could be grouped into those that dominated the mesophilic low pH initial phases (sequences similar to genera Candida, Pichia and Dipodascaceae) and those found mostly or exclusively in the thermophilic phase (sequences clustering to Thermomyces, Candida and Rhizomucor), but a few were also present throughout the whole process., Conclusions: The community composition was found to vary between suboptimally and optimally operating processes. In addition, there were differences in fungal communities between processes of industrial and pilot scale., Significance and Impact of the Study: The results of this study reveal the fungal diversity with molecular methods in industrial composting process. This is also one of the first studies conducted with samples from an industrial biowaste composting process.

Actinobacteria are believed to play a major role in organic matter degradation and humification processes in composts. In this study, the effects of different temperature regimes on the succession of Actinobacteria populations during composting were investigated in a laboratory reactor. Phospholipid fatty acid (PLFA) was used to investigate quantitative changes in the overall microbial biomass and community structure, and in the size of Actinobacteria populations. Qualitative changes were determined using PCR-DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rRNA genes with Actinobacteria-specific primers. The peak in total microbial biomass was roughly twice as high and delayed in trials where the maximum temperature was 40 degrees C, compared to those where it was 55 or 67 degrees C. There was a shift from members of Corynebacterium, Rhodococcus and Streptomyces at the onset to species of thermotolerant Actinobacteria in the cooling phase, e.g. Saccharomonospora viridis, Thermobifida fusca and Thermobispora bispora. In conclusion, temperature was an important selective factor for the development of Actinobacteria populations in composts, and they constituted a substantial part of the community in the later compost stages.

Two genes involved in protein secretion, encoding the Rab protein YPT1/YPTA and the general fusion factor NSFI/NSFA, were characterized from two filamentous fungi, Trichoderma reesei and Aspergillus niger var. awamori. The isolated genes showed a high level of conservation with their Saccharomyces cerevisiae and mammalian counterparts, and T. reesei ypt1 was shown to complement yeast Ypt1p depletion. The transcriptional regulation of the T. reesei ypt1, nsf1, and sar1 genes, involved in protein trafficking, was studied with mycelia treated with the folding inhibitor dithiothreitol (DTT) and with brefeldin A, which inhibits membrane traffic between the endoplasmic reticulum and Golgi complex. The well-known inducer of the yeast and T. reesei unfolded protein response (UPR), DTT, induced the nsf1 gene and the protein disulfide isomerase gene, pdi1, in both of the experiments, and sar1 mRNA increased in only one experiment under strong UPR induction. The ypt1 mRNA did not show a clear increase during DTT treatment. Brefeldin A strongly induced pdi1 and all of the intracellular trafficking genes studied. These results suggest the possibility that the whole secretory pathway of T. reesei could be induced at the transcriptional level by stress responses caused by protein accumulation in the secretory pathway.

The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

We have isolated the birch homologue (BP8) for the carrot embryogenic gene DC8 by heterologous hybridization. The birch BP8 gene encodes a putative protein of 53 kDa, showing 52% sequence identity with the DC8 gene at the amino acid level. The putative BP8 protein contains 20 repeats of 11 amino acids and thus belongs to the group of LEA proteins isolated from such plants as carrot, cotton and wheat. Northern hybridization of mRNA isolated from birch cells representing different stages of somatic embryogenesis and non-embryogenetic material with a PB8 probe gave no signals, suggesting a low expression level of the BP8 gene.