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Researchers turn to young poplars for speedy biofuel
 Are petite poplars the future of biofuels? UW studies say yes

Michelle Ma UW News

In the quest to produce affordable biofuels, poplar trees are one of the Pacific Northwest’s best bets — the trees are abundant, fast-growing, adaptable to many terrains and their wood can be transformed into substances used in biofuel and high-value chemicals that we rely on in our daily lives.

But even as researchers test poplars’ potential to morph into everything from ethanol to chemicals in cosmetics and detergents, a commercial-scale processing plant for poplars has yet to be achieved. This is mainly because production costs still are not competitive with the current price of oil.

A University of Washington team is trying to make poplar a viable competitor by testing the production of younger poplar trees that could be harvested more frequently — after only two or three years — instead of the usual 10- to 20-year cycle. These trees, essentially juveniles compared with fully grown adults, are planted closer together and cut in such a way that more branches sprout up from the stump after each harvest, using the same root systems for up to 20 years. This method is called “coppicing,” and the trees are known as poplar coppice.

This video shows the speed with which young poplar can be harvested. Video provided by Rick Stonex/GreenWood Resources, Inc.

The team is the first to try converting the entire young tree — including leaves, bark and stems — into bio oil, a biologically derived oil product, and ethanol using two separate processes. Their results, published this summer in two papers — one in ACS Sustainable Chemistry & Engineering and the other in Biotechnology for Biofuels — point to a promising future for using poplar coppice for biofuel.

“Our research proved that poplar coppice can be a good option to meet the cheap, high-volume criteria of biofuel feedstock,” said lead author Chang Dou on both papers, a doctoral student in the UW’s Bioresource Science and Engineering program. “Our findings are significant for the future biofuel industry, and the ultimate goal is to make poplar coppice biofuel a step closer to the pump.”

Poplar woodchips from older trees have been the focus of most research, mainly because wood parts contain the highest concentration of sugar, which is important for making ethanol and chemicals. Earlier studies show that poplar woodchips are a viable biofuel source, but costs still don’t pencil out, especially since trees are cut just once every 10-plus years. Additionally, other tree parts go to waste when only the trunk is used, making the process more inefficient and wasteful.

However, if poplar were planted close together like an agriculture crop, and whole trees were harvested on a much quicker cycle, it could make sense from a cost perspective and offer a short return on investment — and be more attractive for farmers.

Alternative fuels must make economic sense, the researchers stress, for biofuels to make a dent in the petroleum-driven market.

“We have the environmental incentives to produce fuels and chemicals from renewable resources, but right now, they aren’t enough to compete with low oil prices. That’s the problem,” said Renata Bura, a UW associate professor in the School of Environmental and Forest Sciences and the senior author.

Bura’s research is part of the Advanced Hardwood Biofuels Northwest project funded by U.S. Department of Agriculture’s National Institute of Food and Agriculture. The project, directed by UW professor Rick Gustafson, is a consortium of universities and industries led by the UW whose goal is to lay the foundation for a Pacific Northwest biofuels and bio-based industry based on poplar feedstock. For this study, trees in Jefferson, Oregon — one of the four study sites — were planted in rows close together in spring of 2012 and harvested less than two years later before the leaves had fallen.

The UW team first tested whether entire young poplar trees could be converted into sugar by a process that uses high temperature, pressure and enzymes to break down the wood materials into sugar. From there, it is possible to make ethanol, acetic acid, lactic acid and other valuable chemicals by fermenting the sugar.

After processing the trees, the researchers found that leaves are poor performers and lowered the overall sugar output, not just because leaves are naturally low in sugar, but they also contain other chemicals that impede the sugar-releasing process. When scaled up to a commercial operation, leaves should be removed and may be used for other purposes, such as feed for animals.

They also tested whole poplar trees from the same plot in another conversion process that uses much higher heat — upwards of 500 degrees Celsius — to transform the tree materials directly to bio oil in a process called “pyrolysis.” Research is underway to convert this dark brown oil to a transportation fuel that resembles gasoline or diesel.

In the experiment, the researchers found that including leaves didn’t make a big difference to the quality of the resulting bio oil. When scaled up, producers could ultimately save time and money by not separating leaves from branches to achieve similar quality oil.

Future poplar production plants could leverage both methods, weighing factors like the current cost of materials or the dollar value of the products being made to determine which method makes more financial sense, Dou explained.

The young poplars used in the study have similar properties to shoots that would sprout from a stump in a true coppicing operation. Using that cutting method, it is possible to harvest trees every two years for up to 20 years without the added effort and cost of pulling up roots, preparing the soil and planting new trees that is required in usual planting regimes.

Ultimately, the researchers say that coppice poplar is likely the best balance of cost and reliability for Pacific Northwest growers to produce biofuel.

“Currently, we are looking at how we can grow poplar for monetized ecosystem services,” Bura said. “In the future, we envision a bio-based industry that will provide multiple environmental benefits, will invigorate rural communities and will serve as a bridge to a fully developed biofuels industry.”

Other co-authors on the papers are Fernando Resende, a UW assistant professor of environmental and forest sciences; Devin Chandler, a UW graduate student in the Bioresource Science and Engineering program; Wilian Marcondes, a UW exchange student from the University of São Paulo-Brazil; and Jessica Djaja, a UW undergraduate student.

The research was funded by a grant from the U.S. Department of Agriculture’s National Institute of Food and Agriculture.

###

For more information, contact Bura at renatab@uw.edu and Dou at changdou@uw.edu.

Grant information:

Agriculture and Food Research Initiative Competitive Grant No. 2011-68005-30407

Output: Researchers at the University of Washington are investigating whether younger poplar trees could be a more viable source of biofuel than older ones. Currently trees are harvested after between 10 and 20 years, but the scientists have experimented with a model that allows them to harvest after only two or three years. The trees are planted closer together and cut to allow new growth to sprout from the same root system, a method known as 'coppicing'. The team then uses the entire tree to make bio oil, without the need to separate the leaves from the branches.


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China starts work on wind tunnel to test hypersonic aircraft
 A J-10 jet fighter performs during the International Army Games on August 13, 2017 in Changchun, Jilin Province of China.

China is building the world's fastest wind tunnel to simulate hypersonic flight at speeds of up to 12 kilometres per second.

A hypersonic vehicle flying at this speed from China could reach the west coast of the United States in less than 14 minutes.

Zhao Wei, a senior scientist working on the project, said researchers aimed to have the facility up and running by around 2020 to meet the pressing demand of China's hypersonic weapon development programme.

"It will boost the engineering application of hypersonic technology, mostly in military sectors, by duplicating the environment of extreme hypersonic flights, so problems can be discovered and solved on the ground," said Zhao, a deputy director of the State Key Laboratory of High Temperature Gas Dynamics at the Chinese Academy of Sciences in Beijing.

The ground tests will significantly reduce the risk of failure when test flights of hypersonic aircraft start.

The world's most powerful wind tunnel at present is America's LENX-X facility in Buffalo, New York state, which operates at speeds of up to 10 kilometres per second – 30 times the speed of sound.

Hypersonic aircraft are defined as vehicles that travel of speeds of Mach 5, five times the speed of sound, or above.

The US military tested HTV-2, a Mach 20 unmanned aircraft in 2011 but the hypersonic flight lasted only a few minutes before the vehicle crashed in to the Pacific Ocean.

In March China conducted seven successful test flights of its hypersonic glider WU-14, also known as the DF-ZF, at speeds of between Mach 5 and Mach 10.

Other countries including Russia, India and Australia have also tested some early prototypes of the aircraft, which could be used to deliver missiles including nuclear weapons.

"China and the US have started a hypersonic race," said Wu Dafang, professor at the school of aeronautic science and engineering at Beihang University in Beijing who received a national technology award for the invention of a new heat shield used on hypersonic vehicles in 2013.

Wu has worked on the development of hypersonic cruise missiles, a near space vehicle, high-speed drones and other possible weapons for the People's Liberation Army.

He said there were a number of hypersonic wind tunnels in mainland China which had helped ensure the high success rate of its hypersonic weapon tests.

The new wind tunnel will be "one of the most powerful and advanced ground test facilities for hypersonic vehicles in the world", said Wu, who was not involved in the project.

"This is definitely good news for us. I look forward to its completion," he added.

More from the South China Morning Post:

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In the new tunnel there will be a test chamber with room for relatively large aircraft models with a wing span of almost three metres.

To generate an airflow at extremely high speeds, the researchers will detonate several tubes containing a mixture of oxygen, hydrogen and nitrogen gases for a series of explosions that could discharge one gigawatt of power within a split second, according to Zhao.

This is more than half of the total power generation capacity of the Daya Bay nuclear power plant in Guangdong.

The shock waves, channelled into the test chamber through a metallic tunnel, will envelope the prototype vehicle and increase the temperature over its body to 8,000 Kelvins, or 7,727 degrees Celsius, Zhao said.

That is nearly 50 per cent hotter than the surface of the Sun.

Output: China is constructing the world's fastest wind tunnel, which will simulate hypersonic flight at speeds of up to 12km per second. At such a speed an aircraft could fly from China to the US in 14 minutes. The project is expected to be in operation by 2020 as China pushes forward its hypersonic weapon development programme. The US tested an unmanned hypersonic flight in 2011 but it lasted only minutes before crashing into the Pacific, while China recently conducted seven successful test flights. "China and the US have started a hypersonic race," said Professor Wu Dafang.


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Scientists use microbiome to develop biofuel enzymes
 Sample collection and enrichment of thermophilic consortia

The sample collection and enrichment procedures have been described previously15. Briefly, compost samples were collected from Jepson Prairie (JP) Organics, located in Vacaville, CA, in 2008. The compost-derived microbial consortium was initially grown aerobically with unpretreated switchgrass and then switched to grow on microcrystalline cellulose (1% wt/vol; Sigma) as the sole carbon source in liquid M9 medium augmented with vitamins. The enrichments were grown at 60 °C and 200 r.p.m. under aerobic conditions in an aerial rotary shaker and serially passed every 14 days with 4% vol/vol inoculum, referred to as passages. Cultivation of the 50 ml culture after passage 80 was scaled at the Advanced Biofuels Process Demonstration Unit, Lawrence Berkeley National Laboratory. A 500 ml culture was inoculated with a 2.5% vol/vol sample from the 50 ml culture and incubated at 60 °C for 14 days at 150 r.p.m. on a rotary shaker. This culture was inoculated into a 19 l bioreactor (Bioengineering USA) to a total volume of 15 l. The 15 l culture was grown at 60 °C, 150 r.p.m. and 0.26 volume gas (sterile air) per volume liquid per minute (VVM) for 14 days. A 400 l bioreactor (ABEC) was inoculated with 7.5 l of culture from the 19 l bioreactor to a volume of 300 l and incubated for 14 days at 60 °C, 150 r.p.m. agitation and 0.25 VVM air sparging. After 14 days, the final fermentation broth was centrifuged using an Alfa Laval Disc Stack centrifuge at 9,000g at 100 l h–1 with 30 min cell discharge interval and 125 kPa back pressure. The clarified broth was collected in the 300 l holding tank bioreactor and the pelleted biomass collected and stored at –80 °C. The supernatant was concentrated through a tangential flow filtration system with a 10 kDa Biomax filter membrane (EMD Millipore). The transmembrane pressure was set at 13 p.s.i. with feed pressure of 30 p.s.i. The concentrated supernatant was freeze-dried under vacuum for 24 h using a lyophilizer (Labconco) and the resulting powder was stored at –20 °C. CMCase and xylanase activities were measured daily for the 15 l and 300 l bioreactor cultivations by removal of samples each day from the bioreactors and the supernatant assayed as described below.

Sequencing, assembly and binning of metagenomics reads

DNA purification from samples extracted from the 15 l and 300 l bioreactors was performed as previously described13. Illumina sequencing (250 bp × 2) of the metagenomic samples was carried out by the Joint Genome Institute (JGI) and performed as previously described40. The sequencing reads of the DNA samples recovered from the 15 l bioreactor (days 1–14 were trimmed using Trimmomatics (with parameter ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36)41 and co-assembled using IDBA-UD42 with the --pre-correction parameter. The 300 l samples (days 4, 5, 7, 10, 12 and 14) were also co-assembled, using the same settings as the 15 l samples. The co-assembled samples for the 15 l and 300 l bioreactor experiments were then binned using MaxBin 2.043 with default parameters, yielding population genomes. The completeness and contamination ratios of these population genomes were assessed using CheckM44. Genomes with >10% of contamination rates were re-binned using MaxBin 2.0 by setting the input contig to the genome file and the input abundance to the extracted abundance files during the whole metagenome binning. The output bins were re-examined using CheckM and the produced bins with higher completeness were chosen to replace the original genome, while the other bins with higher levels of contaminants were discarded. The most likely taxonomic ranks of the recovered genomes were predicted by searching the predicted proteins against the NCBI non-redundant (NR) database, collecting and processing the hits using the least common ancestor (LCA) algorithm proposed by MEGAN445 and assigning the most probable taxonomic rank to the recovered genomes according to the LCA results. GH genes present in the recovered population genomes were identified by using Prodigal46 and then annotated using dbCAN47.

DNA was isolated from a 50 ml aerobic shake flask culture with a bacterial consortium that was adapted from green waste compost obtained from Newby Island Sanitary Landfill in Milpitas, CA, by growth on crystalline cellulose. The enzymatic activities and microbial community membership of this consortium have been described previously13. Metagenomic sequencing was performed by the JGI as described above, and the sequenced reads were assembled as previously described40. Population genomes were recovered by automated binning with Maxbin43 and checked for completeness and contamination with CheckM44.

16S and 23S rRNA gene analysis

A partial 16S rRNA gene (706 bp) was recovered from the ‘Ca. R. cellulovorans’ metagenomic bin. This fragment was used to identify a nearly full-length 16S rRNA gene (1,597 bp; 99.7% identical JGI gene ID Ga0074251_1085371) that was recovered from the initial assembled metagenome (JGI taxon ID 3300005442) obtained for this consortium when it had been adapted to switchgrass40. This observation indicated that ‘Ca. R. cellulovorans’ was present in the consortium when it was adapted to switchgrass, before transferring the consortium to grow on microcrystalline cellulose. Sequences from three clones (GenBank accessions KC978751, KC978760 and KC978763) were >99% identical to portions of the full-length rRNA ‘Ca. R. cellulovorans’ sequence. These clones were recovered from DNA samples isolated from a time series (14 days) of an adaptation of compost from Newby Island Landfill (Milipitas, CA, USA) to grow with microcrystalline cellulose as the sole carbon source at 60 °C13. As described above, a partial genome was recovered from this cultivation that was >99% identical at the amino acid level to the ‘Ca. R. cellulovorans’. A partial 23S rRNA gene (1,444 bp) was recovered from the ‘Ca. R. cellulovorans’ metagenomic bin. The 16S and 23S rRNA gene sequences were aligned using MUSCLE48 trimmed using Gblocks49, and the phylogenetic tree was constructed using MEGA550 with the Tamura–Nei model. Bootstrap values were calculated with 1,000 replicates.

Reconstruction of ‘Ca. R. cellulovorans’ GH gene cluster

Six small contigs were identified in the ‘Ca. R. cellulovorans’ population genome, which contained partial genes containing a catalytic domain (GH9, GH48, GH6/5, 2×GH10, AA10) linked to at least one CBM3. The clustering of these genes was confirmed by PCR amplification of DNA isolated from the cellulolytic consortium. PCR primers (Supplementary Table 8) were designed using the CLC Main Workbench (Qiagen) and PCR products were cloned into pJET1.2/blunt Cloning Vector (Fermentas) and sequenced with an ABI system according to the manufacturer’s instructions. Assembly of gene sequences into a gene cluster and annotation of genes was performed with the CLC Main Workbench and checked for chimerae using the Bellerophon algorithm51.

Phylogenetic analysis

Alignments of protein sequences were performed using the CLUSTALW multiple alignment accessory application in the CLC Main Workbench (Qiagen). In brief, phylogenetic trees were constructed using the CLC Main Workbench applying the maximum likelihood method based on the Whelan and Goldman protein substitution model52. Bootstrap values were calculated with 1,000 replicates.

To build the concatenated protein tree, genes were first searched against the PFAM profiles53 using HMMER354. Genes with PFAM annotations that appear once and only once across all involved genomes were aligned separately using MUSCLE48. After the alignments were concatenated and trimmed using Gblocks49, the concatenated maximum-likelihood protein tree was constructed using MEGA550 with the JTT (Jones, Taylor, Thorton) model. Bootstrap values were estimated with 1,000 replicates.

Protein purification

Lyophilized supernatant (170 mg) obtained from the 300 l cultivation was dissolved in 5 ml H 2 O and passed through a 0.2 µm filter. The supernatant was desalted by dialysis against the buffer (20 mM Tris, pH 8.0) for 24 h with three buffer changes, followed by a 30 ml NaCl gradient fractionation (0–2 M NaCl) using a 5 ml HiTrap Q HP column on an ÄKTA Protein Purification System (GE Healthcare).

Cellulases in the supernatant from the 300 l cultivation were also enriched by binding to phosphoric acid swollen cellulose (PASC), an adaptation of a procedure previously described for cellulosome purification from Clostridium thermocellum 26. Briefly, 250 mg of lyophilized PASC produced from Avicel PH-105 was added to 500 mg of supernatant dissolved in 10 ml H 2 O and mixed at room temperature with a magnetic stir bar for 30 min. After a binding step at 4 °C for 2 h, the amorphous cellulose was centrifuged for 10 min at 3,000g and rigorously washed for 6 cycles with 25 ml reaction buffer (25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0). Washed PASC was resuspended in 10 ml reaction buffer and transferred into dialysis membranes (SnakeSkin and Slide-A-Lyzer; Fisher Scientific) with a 3.5–10 kDa cutoff and dialysed at 60 °C against 4 l reaction buffer at 55–60 °C for up to 48 h with three buffer exchanges per day to prevent possible product inhibition. Dialysis membranes used in this study consisted of regenerated cellulose and were destabilized by cellulases of the substrate, and thus needed to be exchanged every 24 h to prevent membrane rupture. The reaction was considered complete after no visible changes to the substrate were observable. By centrifugation for 20 min at 3,000g the enrichment was split into residual biomass (in the pellet) and the affinity digestion protein fraction (in the supernatant, AD).

Measurement of protein concentration and GH activity

Protein concentrations were determined using the bicinchoninic (BCA) assay (Pierce BCA Protein Assay Kit, Thermo Scientific) method using a 96-well plate (200 μl reaction volume) with bovine serum albumin as the standard. CMCase and xylanase activity assays were conducted as described previously55. Enzyme activity units (U) were defined as µmol of sugar liberated per min. Enzyme activity units for supernatant preparations were calculated as U per ml of supernatant volume. CMC activity units of purified heterologously expressed proteins were reported as U per mg, representing specific activity measurements.

Soluble substrates (p-nitrophenyl (pNP)-labelled) with cellobiohydrolase (pNPC), β-d-dglucosidase (pNPG), β-D-xylosidase (pNPX) and α-l-arabinofuranosidase (pNPA) activities were used to determine enzyme activities on their respective substituents56. The p-nitrophenyl substrate (90 μl) was incubated with 10 μl of diluted enzyme, incubated for 30 min, and quenched with 50 μl of 2% cold sodium bicarbonate. The absorbance of released p-nitrophenyl was measured at 410 nm. Activities using p-nitrophenyl substrates were calculated as U ml−1.

Saccharification of cellulose substrates

Saccharifications were performed in the presence of 2% (wt/vol) Avicel (Sigma) and PASC. Each mixture was prepared in 50 mM MES, pH 6.0 with 10 mg protein per g glucan in biomass to a final volume of 625 µl in a 2 ml screw-cap vial. Saccharifications were carried out at 70 °C in a shaker for 72 h, with 50 µl samples taken every 24 h. All hydrolysates were collected via centrifugation at 21,000g for 5 min and 0.45 μm filtered to remove large biomass particles prior to sugar analysis. After filtration, samples were kept frozen at −20 °C and thawed before analysis. Glucose concentrations were measured on an Agilent 1200 Series HPLC system equipped with an Aminex HPX-87H column (Bio-Rad) and refractive index detector. Samples were run with an isocratic 4 mM sulfuric acid mobile phase. Sugar concentrations were determined using standards containing cellotriose, cellobiose, glucose, xylose and arabinose.

PAGE and zymograms

SDS–PAGE was performed with 8–16% Protean TGX protein gradient gels (Bio-Rad) with the Tris-glycine-SDS buffer57. Blue Native (BN)–PAGE58 was performed with 3–12% NativePAGE Bis-Tris protein gradient gels (Thermo Scientific) in presence of 0.02% Coomassie Blue G-250. For subunit analysis of native complexes, individual lanes from the BN–PAGE were excised, incubated in 2% SDS and 160 mM dithiothreitol (DTT), and denatured at 95 °C for 10 min, unless otherwise indictated (Fig. 3e). Proteins were separated with 8% polyacrylamide gels, which were hand cast. Protein bands were stained with SimplyBlue SafeStain Coomassie Blue dye (Thermo Scientific) according to the manufacturer’s instructions.

Protein bands with activity on CMC and xylan were visualized using modification of the zymogram technique, as described previously15. Gels were incubated in 2% wt/vol CMC or 2% wt/vol birchwood xylan solutions followed by incubation at 60 °C for up to 2 h in reaction buffer (25 mM MES, pH 6.0). In-gel enzymatic activities were visualized by incubating gels with a 0.5% Congo Red solution for 15 min and subsequent multiple washing steps with 20% NaCl.

Glycosylation analysis

Protein glycosylations were visualized in-gel by the periodic acidic Schiff stain27 using a Pierce Glycoprotein Staining Kit (Thermo Scientific) according to the manufacturer’s instructions.

N-glycan analysis was performed as described previously59. However, no N-linked glycans were detected. Total glycosyl compositional analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis60. O-linked glycans were released by β-elimination and permethylated59. The permethylated O-linked glycans were analysed by matrix assisted laser desorption/ionization-time of flight (MALDI–TOF) and electrospray ionization tandem mass spectrometry (ESI MS/MS)61 and gas chromatography/mass spectrometry (GC/MS) for linkage analysis62.

Proteinase K digestion

The E. coli-expressed CelA and CelC and the AD fraction were digested at 50 °C for 60 min in reaction buffer (20 mM Tris-HCl, 400 mM NaCl and 0.3% SDS, 5 mM EDTA containing 75 µg of respective enzyme and 3.75 µg proteinase K). After heat inactivation of proteinase K at 95 °C, the reaction mixture was analysed by SDS–PAGE (8–16% gradient).

Proteomic analysis

Proteins were digested from SDS–PAGE gels as previously described63. Samples were analysed on an Agilent 6550 iFunnel QTOF mass spectrometer coupled to an Agilent 1290 UHPLC system, as described in ref. 64. Briefly, peptides were loaded onto an Ascentis Express Peptide ES-C18 column (10 cm length × 2.1 mm internal diameter, 2.7 µm particle size; Sigma Aldrich) operating at 60 °C and at a flow rate of 400 µl min–1. A 13.5 min chromatography method with the following gradient was used: the initial starting condition (95% Buffer A (0.1% formic acid) and 5% Buffer B (99.9% acetonitrile, 0.1% formic acid)) was held for 1 min. Buffer B was then increased to 35% in 5.5 min, followed by an increase to 80% B in 1 min, where it was held at 600 µl min–1 for 3.5 min. Buffer B was decreased to 5% over 0.5 min, where it was held for 2 min at 400 µl min–1 to re-equilibrate the column with the starting conditions. Peptides were introduced into the mass spectrometer from the UHPLC by using a Dual Agilent Jet Stream Electrospray Ionization source operating in positive-ion mode. The source parameters used include a gas temperature of 250 °C, drying gas at 14 l min–1, nebulizer at 35 p.s.i.g, sheath gas temp of 250 °C, sheath gas flow of 11 l min–1), V Cap of 5,000 V, fragmentor V of 180 V and OCT(octopole) 1 RF (radio frequency) V pp of 750 V. The data were acquired with Agilent MassHunter Workstation Software, LC/MS Data Acquisition B.06.01 (Build 6.01.6157). The resultant data files were searched against a data set containing reconstructed population genomes from the 300 l bioreactor, with common contaminants appended, with Mascot version 2.3.02 (Matrix Science), then filtered and refined using Scaffold version 4.6.1 (Proteome Software).

Heterologous protein expression

Constructs for the CelABC genes were obtained both by PCR amplification from metagenomic DNA with specific primers (Supplementary Table 8) and synthesis of codon-optimized versions for expression in E. coli (Gen9). Genes were cloned into the modified bacterial expression vector pET39b(+) vector with a T7/lac promoter and a TEV-cleavable C-terminal 6xHis tag but lacking the DsbA secretion sequence (Novagen) using Gibson assembly65. All reagents were purchased from New England Biolabs. The desired genes without their signal sequences and the expression vector were PCR-amplified, DpnI-digested and incubated with 1× Gibson assembly Master Mix for 15 min at 50 °C. The product was then transformed into chemically competent E. coli DH10α cells for storage and for heterologous protein expression into chemically competent E. coli BL21 (DE3). Starter cultures (50 ml) of E. coli BL21 (DE3) harbouring plasmids were grown overnight in LB medium containing 25 μg ml–1 kanamycin at 37 °C and shaken at 200 r.p.m. in rotary shakers. Expression was performed in Terrific broth with 2% glycerol, 25 μg ml–1 kanamycin and 2 mM MgSO 4 . Starter cultures were used to inoculate 1 l of expression medium in a 2 l baffled Erlenmeyer flask and incubated at 18 °C while shaking (200 r.p.m.), and induced with 500 µM isopropyl β-d-thiogalactopyranoside (IPTG). Following induction, cultures were again incubated at 18 °C. At 22 h, cultures were centrifuged at 15,500g for 30 min. Cell pellets were resuspended in 25 ml lysis buffer (50 mM NaPO 4 , 300 mM NaCl, 5 mM imidazole; pH 7.4) and homogenized with an EmulsiFlex-C3 instrument (Avestein). After incubation at 60 °C for 30 min, lysates were collected via centrifugation at 75,000g for 30 min and 0.45 μm filtered to remove large particles before purification. Polyhistidine-tagged proteins were purified on Cobalt-NTA resin (Thermo Scientific). To cleave the 6xHis Tag, 1 g purified protein was incubated with 50 mg His-tagged TEV-protease and simultaneously dialysed against 4 l reaction buffer (50 mM NaPO 4 , 300 mM NaCl; pH 7.4) for 24 h and three reaction buffer exchanges. After a second purification step via Cobalt-NTA resin, the flow-through fractions contained the purified and untagged proteins. Proteins were stored at 4 °C until ready for use. The proteins were >90% pure as visualized by SDS–PAGE (Fig. 4c).

Life Sciences Reporting Summary

Further information on experimental design and reagents is available in the Life Sciences Reporting Summary.

Data availability

Metagenomic sequencing data can be accessed at the JGI IMG website (http://img.jgi.doe.gov/) or the JGI Genome Portal (http://genome.jgi.doe.gov/), and the specific IMG genome IDs are listed in Supplementary Table 12. The draft genome sequence for ‘Candidatus Reconcilbacillus cellulovorans’ has been deposited at GenBank (MOXJ00000000). The gene sequences and plasmid constructs for the ‘Ca. Reconcilbacillus’ cellulases CelA (JPUB_007824), CelB (JPUB_007826) and CelC (JPUB_007828) are available from the public version of the JBEI Registry (https://public-registry.jbei.org) and are physically available from the authors and/or Addgene (http://www.addgene.org) upon request.

Output: Scientists from the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory, have used microbiomes to create stable new enzymes which could be used in the production of biofuels and bioproducts. According to a report in the journal Nature Microbiology, the team cultivated microbes in communities, as would occur in the wild, yielding cellulases that had "remarkably robust carbohydrate-protein complexes", and managed to maintain the microbial community for more than three years.


Instruction: create a high quality summary of the given input

Input:
Scientists use microbiome to develop biofuel enzymes
 Newly published research has demonstrated the importance of microbial communities as a source of stable enzymes that could be used to convert plants to biofuels.

The research was carried out by scientists from the Department of Energy’s Joint BioEnergy Institute (JBEI) based at Lawrence Berkley National Laboratory. The findings have been published in the journal Nature Microbiology, and report on the discovery of new types of cellulases - enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts, according to a JBEI statement.

Unlike traditional methods, the cellulases were cultured from a microbial community or microbiome. Usually, isolated organisms are used to obtain enzymes.

“Here we’re cultivating an entire community of microbes to access enzymes that we couldn’t get from isolates,” said study principal investigator Steve Singer, senior scientist in Berkeley Lab’s Biological Systems and Engineering Division and director of Microbial and Enzyme Discovery at JBEI.

“Some microbes are difficult to culture in a lab. We are cultivating microbes living in communities, as they occur in the wild, which allows us to see things we don’t see when they are isolated. This opens up the opportunity to discover new types of enzymes that are only produced by microbes in communities.”

It was revealed that the bacterial population Candidatus Reconcilibacillus cellulovorans yielded cellulases that were arranged in ‘remarkably robust carbohydrate-protein complexes’, a structure never before observed in isolates.

“The enzymes persist, even after a decline in bacterial abundance,” said Singer. “We kept the microbial community cultivation going for more than three years in the lab.”

This stability makes the new cellulose complexes attractive for applications in biofuels production, the authors say. In particular, it gives the cellulases an advantage over alternatives that degrade more rapidly at high tremperature.

Industrial scale

JBEI scientists collaborated with researchers from the Advanced Biofuels and Bioproducts Process Demonstration Unit (ABPDU) at Berkeley Lab, a scale-up facility established by DOE to help accelerate the commercialisation of biofuels research discoveries, to determine whether the enzyme production could be scaled up for industrial applications.

While the JBEI scientists were able to produce 50 millilitre samples at the DOE Bioenergy Research Centre, in just six weeks the scientists at ABPDU scaled the cultures to a volume 6,000 times larger, 300 litres, in industrial bioreactors.

Sebastian Kolinko, a JBEI postdoctoral researcher, is the study’s lead author.

Output: Scientists from the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory, have used microbiomes to create stable new enzymes which could be used in the production of biofuels and bioproducts. According to a report in the journal Nature Microbiology, the team cultivated microbes in communities, as would occur in the wild, yielding cellulases that had "remarkably robust carbohydrate-protein complexes", and managed to maintain the microbial community for more than three years.


Instruction: create a high quality summary of the given input

Input:
Chinese quietly investing directly into India at increasing rate
 Chinese President Xi Jinping and Indian Prime Minister Narendra Modi visit Daci'en Temple on May 14, 2015 in Xi'an, China. Sheng Jiapeng | CNSPHOTO | VCG | Getty Images

If you go by official Indian government figures, Chinese direct investments into the country this century hit a paltry $1.6 billion in March 2017. That's a weak showing compared to President Xi Jinping's 2014 promise to spend $20 billion over five years in India, one of the fastest growing Asian economies. But while there's yet to be big bang investments accompanied by political fanfare, the Chinese have steadily and quietly been investing in Indian companies over the past few years. In fact, people in India who work closely with Chinese businesses estimate that China's investments into the country could be at least five times greater than official numbers. "Chinese investments have doubled in the last two years. I have no reason to doubt that it will not continue as they have already tasted blood. If you are a Chinese company today with a limitless amount of capital and you look at the whole world and ask 'where is the big bet you can play?' the answer is India," said Santosh Pai, a partner at Link Legal, a law firm with offices throughout India. For his firm, Chinese clients are the growth drivers, and since 2011 he has helped about 150 of them get a foothold in the Indian market. Typically, investments come in from Chinese-owned companies based in the U.S., Singapore or elsewhere in the world, and therefore cannot be classified as "Chinese" investments. Also, official data only tracks investments made by companies based in mainland China and not even those out of Hong Kong and Macau, experts said.

Low key, high returns

While businesses are happy to get Chinese money, it is in no one's interest to publicise those investments given the history of Indo-China relations. The two countries have fought a war and have regular skirmishes on their shared border. "If a Chinese company says, 'We have entered the Indian market,' they will attract more scrutiny. If Indian companies say, 'We have Chinese investors,' they may lose some goodwill. It is not a conspiracy to hide these things, but the question is: Who does it benefit to stand on the roof and shout out?" Pai told CNBC. A case in point is the recent investment of $1.1 billion by Shanghai Fosun Pharmaceutical Group for a 74 percent stake in India's Gland Pharma. The Chinese major that had struck a deal in July last year to buy an 86 percent stake in the Indian generic injectable drugmaker, reduced its stake size in a bid to save the stalled deal. The deal had raised concerns among some in the Indian government, Reuters reported. India allows foreign investment of up to 100 percent in its pharmaceutical sector, anything above 74 percent requires government clearance. "Investments that are not administered or influenced by government perception are pouring in," Aravind Yelery, assistant director at the New Delhi based Institute of Chinese Studies, told CNBC. "The Indian state governments are wary of the Chinese, so they are now doing [mergers and acquisitions] and [joint ventures] in the private sector. No mainstream, huge infrastructure investments."

Boosting start-up morale

Instead, Chinese investors are coming into India with bite-sized deals of as little as $1 million for start-ups. The big spenders on that front have included Tencent and Alibaba . The two have invested $1.2 billion and $650 million, respectively, in venture capital and private equity deals over the past two years, according to data from tracking firm Venture Intelligence. That influx of funds has had a notable result in the Indian start-up community. "The Chinese are boosting morale. There is a sudden change in the perception of Indian investors as well. If the Chinese are putting money into Indian start-ups, so will I, is the feeling," said Rishabh Lawania, co-founder of Xeler8, a start-up bought by a Chinese fund in January this year. Xeler8 was a data mining company, but after the buyout it functions as a venture capital fund and incubator for start-ups. "The Chinese are here with deep pockets. It is not uncommon for them to write a million dollar check for a first stage investment. An Indian angel investor is not going to do that. He will invest one fourth that amount," said Sunil Kalra, a veteran investor in Indian start-ups. About $3.5 billion has been invested in companies less than 10 years old so far in 2017, according to Venture Intelligence. The Chinese have contributed a little less than 10 percent of that.

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Output: Chinese direct investment is flooding into India under the radar, with indications the deals are at least five times greater than official figures show. Investments are going unnoticed because they are relatively small or are coming from Chinese-owned units headquartered outside mainland China. "Chinese investments have doubled in the last two years. I have no reason to doubt that it will not continue," said Santosh Pai at Indian law firm Link Legal. Aravind Yelery at the Delhi-based Institute of Chinese Studies said "investments that are not administered or influenced by government perception are pouring in".


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Zocdoc introduces in-app Insurance Checker feature
 The complicated language commonly found in health plans makes it hard for consumers to understand the coverage their plan provides, especially when it comes to determining what physicians are in-network. More than 50% of insured Americans say they have a difficult time figuring out what their insurance will cover, and 77% would like an easier way to ensure their doctor is in-network when booking appointments, according to a survey by Kelton Global.

To remedy the problem, healthcare technology provider Zocdoc developed Insurance Checker, a new feature for its Patient-Powered Search app that helps patients find a doctor. Insurance Checker enables patients to understand the coverage under their health plan by providing such information as whether their doctor is in-network and the patient’s eligibility.

In the first week after launching Insurance Checker last month patients uploaded more than 10,000 images of insurance cards.

Such information is important to consumers when making decisions about their care, as 22% of insured Americans would skip going to the doctor if they were unable to book an appointment with an in-network doctor, and 17% would go to the emergency room for treatment instead, says the Kelton Global survey, which was sponsored by Zocdoc.

To get information about their health plan, consumers open the Zocdoc app, select Insurance Checker and snap a photo of their health insurance card using their smartphone. Insurance Checker scans the photo to gather information on the card such as name of the insurance carrier, plan type and member identification number. Combining information from the card with information from the eligibility check and information doctors provide, Insurance Checker can identify in-network physicians for the patient. Insurance Checker uses artificial intelligence to learn how to match information from an image of a patient’s insurance card with information about his health plan, Zocdoc says.

“With more consumers having high deductible plans, staying in network when booking a doctor’s appointment is important to patients,” says Ruth Sylvia, senior product manager for Zocdoc.

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Insurance Checker can also verify patient eligibility, and, if the information is available, estimate the patient’s co-pay. Confirming benefits eligibility prior to booking a doctor’s appointment ensures patients will be covered for a doctor’s visit. Of the patient’s surveyed by Kelton, 20% booking an appointment with a new doctor were turned away because the doctor didn’t accept their insurance, the survey says. In addition, 16% of insured Americans making an appointment with a new doctor did so not knowing if their insurance would cover the visit.

Confirming patient eligibility in advance of booking an appointment also benefits physicians, as doctors’ office administrators typically contact a new patient ahead of an appointment to ensure the patient’s insurance information is valid. Once an appointment is booked, Insurance Checker shares patient eligibility with the doctor’s office. Insurance Checker also saves the scanned image of a patient’s insurance card to his account for use with future appointments.

Confirmation of patient eligibility is provided by Eligible Inc., a developer of application programming interfaces for collecting medical eligibility, claims, and payments data from insurance companies.

In the first week after launching Insurance Checker last month patients uploaded more than 10,000 images of insurance cards, Sylvia says. The cost for physicians to subscribe to the Zocdoc app is $3,000 per doctor annually. The app, which is available for iOS and Android, is free to patients.

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Output: US medical app Zocdoc, which enables consumers to make doctors appointments, has added a new feature, dubbed Insurance Checker. The in-app service allows prospective patients to check if a doctor or a treatment is covered under their health insurance plan. More than three-quarters of Americans said they need greater clarity on which doctors and medical services are in their insurance networks, according to a survey sponsored by the firm. Additionally, 22% of survey respondents said they would skip treatment if they could not find an in-network provider.


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$1 gift cards aim to make the stock market accessible to all
 Lele's privately-held company is a clinic in making investing more accessible: it makes gift cards, sold online and at various physical locations, that function as fractional shares of stocks.

"[In] the last eight years, the tide has been rising. [The] stock market has been doing well, but most of us don't feel rich, right? Because our ships have been in dry dock. They haven't been in the water," Lele told " Mad Money " host Jim Cramer in an exclusive interview on Monday. "And here, to get into the market, I think the easiest point of access is to invest in something you know, something that you love."

Recent statistics show the number of Americans invested in stocks declining , but Stockpile Co-founder and CEO Avi Lele told CNBC his fractional share trading platform can help change that.

Consumers can also open a brokerage account with Stockpile's mobile app and start investing at 99 cents a trade. The app offers investing advice so newcomers can learn basic investing rules.

"People are told all the time, 'Start early, diversify and do it for the long haul,' but it's really hard to start early when you're young because you don't have a lot of money and you certainly don't know anything about the market," Lele said. "They don't teach it to you in school and you can't walk into a traditional brokerage saying, 'Hey, I've got $50 bucks, how does the market work?'"

The CEO said he ushered his own children into learning about investing with Stockpile when he realized they weren't interested in investing through traditional firms.

Lele had set up custodial accounts for them through a traditional brokerage firm, but he was the only account holder and just couldn't get his kids interested, he said.

But with Stockpile, "they had their own login. They could check in on their stocks, they could buy things that they knew instead of mutual funds that were hard to [grasp] and they could place trades that came to my phone for approval," Lele said. "All of a sudden, an engaging experience was born. Just a few hundred dollars, but they had skin in the game."

During a visit to his dentist, Lele even thought up a Halloween promotion that he thought would spur young people's interest in his platform.

"I was at my dentist a few weeks ago, and dentists hate giving out candy," the CEO said. "And I told him about the company and he said, 'Wouldn't it be neat if I could give out $1 stock cards to the trick-or-treaters?' And so I'm sitting there looking over here and saying, 'Well, Nike kind of goes well with Amazon, orange and black, you want to give those out?'"

Lele's central idea is that people, including disillusioned millennials, can warm to investing if they're buying shares in companies they know and love at affordable prices instead of the lucrative, but astronomically priced stock market.

"If you're an Apple fan, start investing in Apple. If you're a Nike fan, if you're a sports fan, invest in Nike. Now, that's the on-ramp," Lele said. "But once you're in, we have a whole series of lessons, many lessons, in our app that are designed to teach you, 'Hey, start diversifying. Start looking at ETFs because they're instant, great diversification. Start investing in maybe other countries' stocks to get some foreign exposure.' So it's a starting point, but then it brings you in and now you're not in dry dock."

Output: US fractional investment platform Stockpile is selling gift cards for as little as 99 cents, with the goal of increasing the number of people engaging with the stock market. Offering consumers access to stocks of well-known companies at a significantly cheaper price than the full shares can encourage more people to learn about investing, said the firm's CEO. Apathetic younger investors are a key audience for Stockpile.


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Financial advisers ponder ways to increase female headcount
 Boosting the number of women in the financial advice business to a level that more equally represents the population may require firms make changes related to hiring, compensation, training and other areas.

It also will require getting more men on board with the effort, according to the comments from a group of about 150 women who gathered Tuesday to discuss ways to increase the ranks of female financial advisers. The group spent three hours brainstorming solutions in a pre-conference session at the Schwab Impact Conference being held in Chicago this week.

“We’re looking for specific actions that we can take back to our firms and communities to further increase the representation of women in this industry,” said Susan Forman, senior vice president and head of marketing for Schwab Advisor Services.

While 51% of the U.S. population are women, only about 15.7% of financial advisers are female, and less than a quarter of Certified Financial Planners are women — amounts that have barely changed in a decade, according to Suzanne Siracuse, publisher of InvestmentNews, who moderated the discussion.

(More: How to encourage the rise of senior women in financial advice)

Recruiting is one of the areas that firms participating in the session said needs to change. At one advisory firm, they have a recruiting team that requires there to be a diverse set of candidates identified before interviews can begin for any position at the firm. That firm also has programs to help encourage more junior employees to experience many different areas of the business to try and find one in which they can particularly thrive.

At another firm, the focus is on work-schedule flexibility, allowing four-day work weeks so advisers at the firm can care for children, or in some cases, provide care for a parent.

Other firms provide women advisers with training and coaching to help with leadership development.

Lisa Poff, director of marketing and communications at Bridgewater Wealth Management, an advisory firm, said compensation structures that are built solely to reward those who are going out and getting business versus those who are taking care of the business is another deterrent for women in this industry.

At Bridgewater, which manages about $1 billion in client assets, the firm actively tries to create an environment that’s supportive to its female advisers.

“We have created a culture of feeling like everyone’s voice is heard,” she said.

Six of its nine advisers are women.

(More: Advice firm carves out unit for women investors)

The group agreed it’s important to get more men involved with the effort to expand the numbers of women in the business.

However, they were not overflowing with examples of successful efforts.

“That’s an area where we need to look for more solutions,” Ms. Siracuse said. “Men need to buy into this and understand the business case for this.”

Output: US financial advisory firms are mulling making changes to their recruitment, compensation and training practices to attract more female employees. Despite more than half of the US population being female, only 16% of financial advisers are women. A number of firms are making sure candidates for open positions come from a diverse pool, while other firms are fostering an environment where employees can have more flexible working hours.


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TalkTalk Dunstone's shareholding falls £33m after loss announcement
 Sir Charles Dunstone saw £33million wiped off the value of his TalkTalk shares after the firm crashed into the red.

The 52-year-old businessman, who owns nearly a third of the internet provider he set up in 2003, is spearheading efforts to revive its fortunes having taken over as executive chairman in February.

But his turnaround plan suffered a bruising setback yesterday TalkTalk posted a £75million loss for the six months to September 30.

Shares tumbled 17 per cent in early trading before closing down 6 per cent or 11.3p at 178.2p.

That cut the value of Dunstone's 294m shares in the company by £33million to £524million.

Output: TalkTalk executive chairman Sir Charles Dunstone saw £33m ($44m) wiped off the value of his shares in the telco after it posted a £75m loss for the six months through September. Dunstone, who owns almost one third of the company, returned to its helm in February to turn its fortunes around. Shares in TalkTalk fell 17% after the loss was announced, reducing the value of Dunstone's holding to £524m.


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Insurers urged to provided equal coverage for mentally ill
 (UNTREATED: This is part of a series on the care of Staten Island's mentally ill. Click here for additional stories in the project.)

STATEN ISLAND, N.Y. -- Despite laws put in place to guarantee equal coverage for mental health treatment, insurance companies are denying mental health claims more often than physical health claims.

In 2015, 29 percent of survey respondents said that their mental health claim was rejected due to the private insurance company saying it was not medically necessary, more than twice the percentage reported for denied medical claims, according to a National Alliance on Mental Illness (NAMI) study.

Fourteen percent of medical claims were denied for the same reason, the study shows.

Affordable Care Act plans had lower total denial rates when compared to other private insurance but mental health claim denials were still almost twice as high as medical claim denials.

Claim denials, as well as low reimbursement rates for services and providers who don't accept insurance create financial burdens for many families, often forcing patients to choose between services they desperately need and services they can afford.

"Middle and working class people can't really afford the financial burden," Fran Zagor, president and CEO of the Staten Island Mental Health Society (SIMHS) told the Advance.

Leslie Moran, senior vice president of the New York Health Plan Association (NYPA), said it's not as simple as comparing mental health to physical health because the term "medically necessary" varies widely for both.

WHAT DOES MEDICALLY NECESSARY MEAN?

Moran said the NYPA has some "misgivings" about the NAMI statistic because physical claims are more defined than mental health in terms of medical necessity.

"Physical health claims -- i.e. a broken leg, doctor's visit for bronchitis, etc. -- are more defined, or more clear cut versus mental health claims where there might be a question about the appropriate number of visits within a time frame (does the patient need two visits per week?)," Moran stated.

The setting where mental health services are provided, such has inpatient versus outpatient treatment, comes in to play as well, she said.

Mental health providers say it is unfair for an insurance company to determine medically necessity for mental health treatment because treatment is more complex and insurance companies do not take into consideration the patient's medical history.

The NAMI report states that there has been little "clarity" about what criteria insurance companies use to determine what is or is not medically necessary.

"In the absence of uniform criteria, insurers have adopted their own standards and have often not been forthcoming about informing beneficiaries about these standards. This in turn has sparked concerns that insurance companies and managed care organizations deny claims for mental health care at far higher rates than other for medical care," NAMI said in the report.

When an insurance company denies a claim, they are required to provide a reason for the denial in writing and include information on how to appeal the process.

Additionally, the NAMI study does not acknowledge or include statistics regarding a person's ability to appeal the claim, Moran said.

"Both internal plan appeals and independent external appeals, and the percentage that are overturned on appeal, is not addressed in this data," she said, adding that it is not "really a fair comparison."

"In approximately 60 percent of disputes about medical necessity, the plan's decision is upheld by an independent external reviewer," she said.

PARITY LAWS IN PLACE

After decades of little to no fair coverage from insurance providers, Mental health parity laws were enacted to require insurance plans to have comparable coverage for mental health, addiction and other medical treatment.

The federal Mental Health Parity and Addiction Equity Act of 2008 prevents group health plans and health insurance issuers that provide mental health or substance use disorder benefits from imposing less favorable limitations on those benefits than on medical/surgical benefits, according to CMS.gov.

This federal law superseded New York state's "Timothy's Law," by replacing the required coverage minimum of 30 days of inpatient and 20 days of outpatient treatment with equal and consistent coverage for mental health treatment.

While parity laws have closed some gaps for mental health coverage, providers say that patient's needs are still not being met.

"The insurance companies say they meet the needs of their members but the reality is, it becomes clear when you're dealing with somebody who has a real issue and people on the insurance panel say 'We can't provide you with services,'" Zagor said.

Moran argued that Zagor's comment shows "provider's bias."

"It often comes down to a question of need versus want. In other words, providers want all the treatment for a patient that they can get the health plan to pay for, even when determination is made that the treatment isn't truly medically necessary," Moran said.

INSURANCE NOT ACCEPTED

While laws have been changed to make treatment more accessible, many treatment providers don't accept private insurance or Medicaid -- due in part to low reimbursement rates and new provider laws -- forcing those seeking treatment to pay out-of-pocket or forgo treatment altogether.

"Parents are calling us desperately looking for places to bring their children. Unfortunately, the rate of reimbursement is not high so we won't sign the contract with the insurance company because of it," Zagor explained. "Except there's nowhere else to refer them too."

She said the SIMHS will work with the families, allowing them to pay off the difference between what the insurance company pays and the cost of services.

Only 55 percent of psychiatrists accept insurance, compared to 88.7 percent of medical physicians in other specialties, a study published by JAMA Psychiatry shows.

Moran acknowledged that there are providers who do not accept insurance, but said it is each provider's decision and does not reflect the on private insurance companies.

The coverage parity law covers deductibles, co-pays, co-insurance, out-of-pocket expenses, annual and lifetime dollar limits, limits on frequency of treatment, number of visits, and other limits on the scope or duration of treatment.

"Health plans have an interest in making sure their members get the right are in the right setting at the right time, which applies to mental health services as well as physical health care. Plans work diligently to comply with the letter and spirit of mental health parity laws that ensure access to needed services," Moran said.

NAMI states that New York is "leading the nation" in terms of coverage parity and enforcement, however, obstacles still exist when trying to find and afford treatment.

"We have the expertise to deal with all of this. We provide a lot of training [for staff], we use evidence-based practices, there are psychiatrists available; in our setting, we have it all but it all goes back to having the right kind of reimbursement," Zagor said.

Output: Despite recently implemented mental health parity laws, sufferers of mental illness in New York State are rarely reimbursed for treatment, even if they have insurance coverage. A number of medical providers don't accept private insurance for treatment, due largely insurers' reluctance to pay out. In 2015, 29% of mental illness suffers said insurers rejected a claim they made following treatment, according to a survey conducted by the National Alliance on Mental Illness.