text:
BAR global bi-weekly crypto regulatory round-up
Report: JCB Developing Blockchain B2B Payment Product
JCB, a Japanese card provider, is developing a blockchain B2B payment offering specifically for netting trades between two parties. JCB is teaming with LayerX, a local company, on the offering, Ledger Insights reported.
Most companies have a one-way partner relationship, but firms that have two-way trade encompass commodity traders, investment companies and maybe the big Japanese firms. Not many particulars of the effort were available, according to the report.
UK Regulator Makes Temporary Register for Crypto Firms Awaiting Permits
The British Financial Conduct Authority (FCA) has launched a temporary program for registering crypto companies that are waiting to obtain approval permits. The move from the financial regulator will enable a large number of crypto businesses to continue their operations beyond the January 10, 2021, deadline for registrations until at least July 9 of next year, reported a crypto news site.
Estonia Has Withdrawn Licenses From Over 1,000 Crypto Companies This Year
According to reports, Estonia has withdrawn more than 1,000 licenses from cryptocurrency companies in 2020, apparently due to the burden of monitoring compliance.
Crypto China: Construction Bank in China Launches First Insurance Policy Paid in Digital Currency
According to reports, China Construction Bank, one of the largest banks in China has started a new use case for the country's much talked about digital yuan by issuing the first insurance policy completely paid in the national digital currency. The digital yuan settled insurance policy was launched in association with ZhongAn Online P&C Insurance.
Cryptocurrency Tries to Fill in During Zimbabwe's Cash Shortages
Since the coronavirus hit the country in March, COVID-19 restrictions have forced depositors to queue early to withdraw only 1,000 ZWL (Zimbabwe dollars) weekly, per central bank regulations, which translated to $3.11 US on Dec. 7. With hyperinflation in the country, that amount goes less and less far each week, reported a crypto news site.
Binance Korea is shutting down just eight months after its launch
According to reports, Binance Korea, a crypto-to-crypto exchange of Binance for Korean users, is shutting down next month. The closure comes just eight months after the platform's launch in April 2020.
India's Uttar Pradesh State Starts India's First Blockchain-Enabled Rooftop Solar Trading Platform
Uttar Pradesh has launched India and South-Asia's first blockchain-enabled 'peer to peer' (P2P) trading platform for rooftop solar power. The Uttar Pradesh Power Corporation (UPPCL), Madhyanchal Vidyut Vitran Nigam Limited (MVVNL), and Uttar Pradesh New and Renewable Energy Development Agency (UPNEDA) are hosting the first-of-its-kind pilot project, involving energy generated from solar rooftop systems installed on buildings in Lucknow, reported a crypto news site.
South Korean Province to Create Youth Blockchain Jobs with New Fund
South Korea's South Jeolla Province has announced plans to spend USD 14m on a job creation project that will see the region develop new roles in the industry 4.0 sector, blockchain including, for promising youngsters. According to a report from Jeonnam Maeil, funding for the project has been secured from central government resources and is part of the wider, Ministry of Public Administration and Security-run, post-coronavirus pandemic economic revival project.
US Floats Long-Dreaded Plan to Make Crypto Exchanges Identify Personal Wallets
According to reports, U.S. cryptocurrency users hoping to transfer their holdings from an exchange to their own personal wallets may need to comply with new know-your-customer (KYC) requirements under a rule proposed by the Treasury Department.
European Union unveils revamp of cybersecurity rules days after hack
According to reports, the European Union unveiled Wednesday plans to revamp the 27-nation bloc's dated cybersecurity rules, just days after data on a new coronavirus vaccine was unlawfully accessed in a hack attack on the European Medicines Agency.
The EU last year recorded around 450 cyber incidents involving European infrastructure, notably in the financial and energy sectors, and the pandemic has highlighted Europe's deep dependence on the internet and exposed security weaknesses.
Hong Kong Grants First License to Cryptocurrency Trading Platform
Hong Kong now has its first regulated cryptocurrency trading platform. The Securities and Futures Commission has just given out the first-ever crypto license to OSL Digital Securities which will soon commence trading for several cryptocurrencies, including bitcoin, reported a crypto news site.
Hong Kong ATMs Must Be Excluded From Coming AML Regulations, Group Says
New anti-money-laundering (AML) regulations need to exclude Hong Kong's bitcoin automated teller machines (ATMs) in order to sustain innovation, according to the Bitcoin Association of Hong Kong, reports the South China Morning Post (SCMP), reported a crypto news site.
US Lawmakers Caution Against Regulations Restricting Use of Self-Hosted Crypto Wallets
Four U.S. lawmakers have sent a letter to Treasury Secretary Steven Mnuchin, warning of the risks of restricting the use of self-hosted cryptocurrency wallets. Their concerns follow reports that the Treasury Department may be on the verge of imposing such strict cryptocurrency regulations aimed at self-hosted crypto wallets, reported a crypto news site.
US Treasury Proposes Sweeping New Disclosure Rules On Bitcoin, Other Cryptos
The U.S. Treasury Department proposed sweeping new rules late Friday (Dec. 18) that the government says would make convertible digital currencies like bitcoin less attractive to criminals engaging in crimes such as ransomware attacks, reported a crypto news site.
Electronic Frontier Foundation: US Government Will Expand Financial Surveillance Through FinCEN's Proposed Crypto Wallet Rules
The Electronic Frontier Foundation (EFF) raised concerns about the strengthening financial surveillance through the proposed crypto regulations announced by the U.S. Treasury's Financial Crimes Enforcement Network (FinCEN), reported a crypto news site.
Germany Legalizes Blockchain-Based Securities
According to reports, new legislation passed by Germany allows all-electronic securities to be recorded on a blockchain. Securities issuers and holders no longer need paper certificates.
While Germany is bullish on blockchain tech, the administration looks less kindly upon crypto and stablecoins.
UK Crypto Businesses Get Reprieve as FCA Extends Consideration Period Until mid-2021
According to reports, UK financial regulators will allow crypto firms with pending registration applications to continue to operate until the middle of 2021. The agency plans to use the extension to clear the applicant backlog as its operations have been hamstrung by the ongoing coronavirus pandemic.
Pro-crypto Elad Roisman appointed new chairman of United States SEC
According to reports, Elad Roisman was earlier appointed by incumbent President Donald Trump as commissioner to the US SEC. He has been with the federal agency since 2018. Many believe the new chair is friendly towards the crypto community. As commissioner Roisman supported Peirce's earlier initiative to include crypto markets in regulatory decisions.
EU rights watchdog warns of pitfalls in use of artificial intelligence
The Vienna-based EU Agency for Fundamental Rights (FRA) urged policymakers in a report issued on Monday to provide more guidance on how existing rules apply to AI and ensure that future AI laws protect fundamental rights, reported a crypto news site.
France to extend digital currency KYC with new regulations
France is introducing a raft of new legislation governing the digital currency sector, including tougher Know-Your-Customer rules for digital currency transactions.
The French finance ministry has reportedly been working on proposals for a stronger regulatory regime, which will see crypto-to-crypto exchanges regulated for the first time, as well as more stringent measures for crypto-to-fiat exchanges and other service providers, reported a crypto news site.
Major Latin American University Launches Specialization Featuring Crypto-Related Topics
One of the biggest and most laureate universities in Latin America approved the launch of a Financial Engineering specialization course, whose modules cover crypto and blockchain-related subjects, marking it a historic landmark across the region's educational sector, reported a crypto news site.


Car dollies are designed to ease and streamline the process of moving a vehicle to and from the showroom or your garage when you are working on it, either for those that cannot or choose not to actually drive the vehicle. For this reason, there are a large number of different types of heavy-duty car dollies on the market – each offering a different size, weight rating, and shape to match the specific needs of your classic truck.
We have long carried and trusted Merrick Machine heavy-duty car dollies for our customers because of their high level of quality and wide range of options for both trucks and other types of vehicles including cars, boats, tractors, and motorcycles. We also carry their line of car dolly accessories.
Merrick Machine heavy-duty car dollies are designed to allow a single person to move very large objects, including a vintage truck. This will allow you to optimize and utilize your work space as efficiently as possible, using the car dolly to move your vehicle to and from that space whenever needed. Whether you are working on your truck only on the weekends and have limited working space or you need to shift positioning in your garage periodically, their auto dollies give you that option.
When using Merrick Machine auto dollies, a dolly is places under each tire of the vehicle. A standard auto dolly is rated to hold up to 1,500lb, meaning a total weight with all four tire of 6,000lb. There are three sizes, however, meaning you can upgrade to the heavy duty auto dollies that can handle up to 2,500lb per dolly (or 10,000lb total).
If you need a new auto dolly set for your classic car or truck, consider a Merrick Machine auto dolly from Carolina Classic Trucks. We offer some of the lowest prices on the market and free shipping on your order. We also sell a number of car dolly accessories. If you don't see the specific size or type of dolly you need in the list below, contact us and ask to speak with a service expert who can help you find one that fits your needs.


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← Maritimes Butterfly Atlas Aviva Campaign – we need your votes!
Maritimes Butterfly Atlas Aviva Campaign – vote now!
The Maritimes Butterfly Atlas has one last chance to qualify for the Aviva Community Fund's semi-final round. So starting today at 1:00PM, we are asking you to vote daily for the project . Voting goes until November 25th. You can vote at http://www.avivacommunityfund.org/ideas/acf18213, and follow our campaign on Facebook at https://www.facebook.com/mbaaviva.
To qualify for Aviva funding we will need votes, votes, and more votes. So, if you could forward this to your networks and friends and encourage them to vote as well as forward it to their networks and friends, that would be a huge help! If you haven't done so already, you must register to vote (you'll be prompted to register when you cast your first vote). Registration is quick and easy, and once you're registered you simply sign in daily to vote.
The Maritimes Butterfly Atlas is hoping to win funds to support the fifth and final year of the project in 2014. To date more than 300 volunteers have participated, and the 17,000 records they've submitted have significantly enhanced the understanding of butterflies in our region. One more year of surveys will further contribute to this already successful project.
Thanks so much for your support! Together we will keep the Maritimes Butterfly Atlas flying high.


text:
@implementation ELRangedOscillator

#pragma mark Object initialization

- (id)initEnabled:(BOOL)enabled minimum:(int)minimum hardMinimum:(int)hardMinimum maximum:(int)maximum hardMaximum:(int)hardMaximum {
  if( ( self = [super initEnabled:enabled] ) ) {
    [self setMinimum:minimum];
    [self setHardMinimum:hardMinimum];
    [self setMaximum:maximum];
    [self setHardMaximum:hardMaximum];
    [self setValue:minimum];

    // NSLog( @"%@ (ranged) value = %d, minimum = %d", self, [self value], minimum );
  }

  return self;

}

- (id)initEnabled:(BOOL)enabled minimum:(int)min maximum:(int)max {
  return [self initEnabled:enabled minimum:min hardMinimum:min maximum:max hardMaximum:max];
}

- (NSString *)description {
  return [NSString stringWithFormat:@"<ELRangedOscillator: %p> value:%d min:%d hard_min:%d max:%d hard_max:%d", self, [self value], [self minimum], [self hardMinimum], [self maximum], [self hardMaximum]];
}

#pragma mark Properties

@synthesize minimum = _minimum;

- (void)setMinimum:(int)newMinimum {
  _minimum = newMinimum;
  _range = [self maximum] - _minimum;

  if( [self value] < _minimum ) {
    [self setValue:_minimum];
  }
}

@synthesize hardMinimum = _hardMinimum;

@synthesize maximum = _maximum;

- (void)setMaximum:(int)newMaximum {
  _maximum = newMaximum;
  _range = _maximum - [self minimum];

  if( [self value] > _maximum ) {
    [self setValue:_maximum];
  }
}

@synthesize hardMaximum = _hardMaximum;

@synthesize range = _range;

#pragma mark Implements ELXmlData

- (id)initWithXmlRepresentation:(NSXMLElement *)representation parent:(id)parent player:(ELPlayer *)player error:(NSError **)error {
  if( ( self = [super initWithXmlRepresentation:representation parent:parent player:player error:error] ) ) {
    BOOL hasValue;

    int min_value = [representation attributeAsInteger:@"minimum" hasValue:&hasValue];
    if( !hasValue ) {
      if( error ) {
        *error = [[NSError alloc] initWithDomain:ELErrorDomain code:EL_ERR_OSCILLATOR_INVALID_ATTR userInfo:[NSDictionary dictionaryWithObjectsAndKeys:@"Ranged oscillator has no or invalid minimum",NSLocalizedDescriptionKey,nil]];
      }
      return nil;
    }

    int max_value = [representation attributeAsInteger:@"maximum" hasValue:&hasValue];
    if( !hasValue ) {
      if( error ) {
        *error = [[NSError alloc] initWithDomain:ELErrorDomain code:EL_ERR_OSCILLATOR_INVALID_ATTR userInfo:[NSDictionary dictionaryWithObjectsAndKeys:@"Ranged oscillator has no or invalid maximum",NSLocalizedDescriptionKey,nil]];
      }
      return nil;
    }

    [self setMinimum:min_value];
    [self setValue:[self minimum]];
    [self setMaximum:max_value];
  }

  return self;
}

- (void)storeAttributes:(NSMutableDictionary *)attributes {
  [super storeAttributes:attributes];

  [attributes setObject:[NSNumber numberWithInteger:[self minimum]] forKey:@"minimum"];
  [attributes setObject:[NSNumber numberWithInteger:[self maximum]] forKey:@"maximum"];
}

@end

redpajama_set_name: RedPajamaGithub


Supplied as a pair of Extendable holdout arms, 5 different lengths for use with the Insta-Lock System. High grade stainless arms are adjustable and designed to secure the inflatable boat in its raised position on davits at 90 deg. to the main vessel. Hold out arm 'Pads' available separately.


Here's our latest roundup of some MENA campaigns that are leading the way with their standout creative strategies, whether for celebrating launches, spotlighting sponsorships, or just jumping into timely conversations.
With a delicious new Italian Angus Burger on offer, Hardee's Arabia (@hardeesarabia) turned to the Video Website Card to drive awareness and online orders. Its witty video caught attention and tempted taste buds, and viewers could click directly through to an ordering page.
Beauty brand Lux Arabia (@LuxArabia) made the most of its Fashion Forward Dubai (@ffwddxb) sponsorship with an In-Stream Pre-Roll Sponsorship on Twitter. @LuxArabia's pre-roll appeared on the Fashion Forward recap video from women's magazine Sayidaty (@sayidatynet), driving awareness of its event sponsorship and reinforcing the brand's association with fashion.
As online shopping destination Noon.com (@noon) launched, it shared the news through Promoted Video on Twitter. Its vivid creative stood out in timelines, conveying excitement and celebration while reflecting the look and feel of the site itself.
App-based car service Careem (@careem) had planned a First View campaign with a Promoted Trend for 24-hour nationwide exposure in Saudi Arabia, its largest market.
When, the night before the First View, it was announced that women in Saudi Arabia would be allowed to drive, @CareemKSA reacted quickly to put its brand at the forefront of the conversation. @CareemKSA made a new video and changed its hashtag مبروك_لنساء_الوطن# so that its campaign fit right in with the topic the whole country was talking about that day.
Chevrolet Arabia (@ChevroletArabia) drove awareness of the all-new Equinox SUV and its features with a First View campaign that included a Promoted Trend as well as a custom emoji.
@ChevroletArabia used Conversational Video, engaging people in answering the question "what worries you most while driving?" When people clicked on an answer, they were able to share a Tweet with their own followers, giving the campaign organic reach — and letting @ChevroletArabia address each worry with relevant information about the Equinox.
Nissan Saudi Arabia (@nissansaudi) also saw an opportunity to join the conversation about women driving. The brand stayed top of mind with a Promoted Tweet that featured the hashtag لسماح_للمراه_بالقياده# and a memorable image.
As the Egypt vs. Congo World Cup Qualifier game approached, Molto Egypt (@MoltoEgypt) wanted to drive conversation that would put its snack brand at the center of the excitement. @MoltoEgypt ran Conversational Cards asking people to predict the game's outcome. As fans shared their predictions, they spread the campaign to their own followers.
Magnum Pakistan (@MagnumPK) hosted a chocolate-themed #MagnumParty17 to spotlight its association with glamour and fashion trends. @MagnumPK spread awareness of the party, and brought its glamour to a wider audience, by Tweeting video before, during, and after the event. Its campaign included Conversational Video and Instant Unlock to keep audiences engaged.


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"The Fed lent freely, but at a low rate, on dodgy collateral"
By Edward Harrison	 On Apr 1, 2011
My view is that the Fed's role should be circumscribed. Nevertheless, the Fed serves an important function in the US and global economy in providing liquidity, as we have witnessed during the crisis. It is the American – and in many respects, the global – lender of last resort. Walter Bagehot wrote an eponymous financial book in the 19th century called Lombard Street, giving advice on finance and banking. The most important and quoted piece of advice Bagehot gave was to the Central Bank, in this case the Bank of England. He told them to "lend freely at a high rate, on good collateral."
This is something the Federal Reserve has failed to do during the credit crisis. The Fed lent freely, but at a low rate, on dodgy collateral. This was true even before Lehman collapsed. See the Wall Street Journal's "Bye, Bye Bagehot" article on Fed lending under the Term Auction Facility from January 2008 for example. The setup of the Fed's Primary Dealer Credit Facility had the clearing banks determine the haircuts banks had to take for taking loans from the Fed. The result has been an inability of market participants, regulators or the wider public to differentiate between the truly insolvent companies and the illiquid. The resulting lack of confidence in the entire financial system and by financial institutions in each other has been damaging to the global economy.
This is the crux of the Fed's problem – and its conduct during the crisis is emblematic of the untenable role of the institution as both regulator and servant of the financial services industry.
–Some Thoughts On The Fed and Bank Bailouts,Dec 2010
The role of a central bank in a crisis is to act as a lender of last resort by helping market participants discriminate between the truly insolvent and the unfortunate illiquid. If the Central Bank lends against good assets at a penalty rate, the truly insolvent financial institution goes bust but the unfortunate illiquid institution gets bailed out. I have been making this point for quite some time.
Nov 2008 (Bloomberg News sues the Fed under Freedom of Information Act): For those of you concerned about the Fed's risky behavior, its ballooning balance sheet, and its acceptance of dodgy collateral, well you may be about to see whether the American democracy can allow this unchecked power to continue without oversight. Bloomberg News has sued the Federal Reserve to force them to reveal what kind of collateral they are accepting in loaning out trillions of dollars to U.S. banks.
Nov 2008 (Bailout Nation): we come to American Express. Apparently, the U.S. Government feels that American Express needs to get on the gravy train as well given that they just granted the company authorization to become a bank holding company. Bank holding companies can borrow at low rates from the Federal Reserve, offering up dodgy collateral like Asset-backed securities in turn. That is why all of the investment banks have become bank holding companies and that is also why the Federal Reserve has been sued by Bloomberg News to find out just what kind of collateral these companies are using.
Jun 2009 (Will banks exiting TARP take back their toxic waste now?): This has not been a liquidity crisis. It is a solvency crisis. The banks are not well-capitalized because the stress tests were just a big charade and an effort to buy these firms time. Moreover, it is painfully obvious that the banks are very much dependent on the government still – or they would be getting their dodgy assets back.
Meanwhile the Bloomberg lawsuit filed under the Freedom of Information Act to uncover what kinds of assets the Fed is now holding as collateral is still ongoing.
So, if you are wondering like me, when the Fed is going to start shaping up its balance sheet or when the Fed is going to come clean about what kinds of dodgy assets it now holds on that balance sheet, you're likely to be waiting for answers for a very long time.
Oct 2010 (Grantham: Night of The Living Fed): [Jeremy] Grantham sees the Fed at the centre of four major events in the post-bubble era: runaway commodity prices, zombie banks that don't lend enough to customers or issue dividends to investors, the foreclosure crisis, and 'the currency wars.' I have written endlessly about the Fed's machinations to pump up asset prices via an asymmetric policy which promotes the accumulation of debt. Like Grantham, I am coming to the conclusion that the Fed needs a lot less power. Even in the circumscribed role which Grantham would permit the Fed, we had to marvel at how it passed out favours to the banks via buying up dodgy collateral for loans at low rates. Moreover, on that very score, let's not forget the secrecy with which the Fed is trying to operate. It has taken a lawsuit to the Appellate courts to get them to come to heel. Of course, the Dodd-Frank legislation passed by Congress and supported by the Obama Administration gives the Fed more not less power.
Well, now the Fed has finally been forced to come clean by the Bloomberg and Fox News lawsuits. And, whocouldanode, they lent $118 billion against dodgy collateral just as I have ben saying.
Headline: Fed Let Brokers Turn Junk to Cash at Height of Financial Crisis
At the height of the financial crisis, the Federal Reserve allowed the world's largest banks to turn more than $118 billion in junk bonds, defaulted debt, securities of unknown ratings and stocks into cash.
Collateral of those asset types made up 72 percent of the total $164.3 billion in market-rate securities pledged to the Fed on Sept. 29, 2008, two weeks after the bankruptcy of Lehman Brothers Holdings Inc., according to documents released yesterday. The collateral backed $155.7 billion in loans on the largest day of borrowing from the Primary Dealer Credit Facility, which was created in March 2008 to provide loans to brokers as Bear Stearns Cos. collapsed.
Did you do the math? $155.7 billion/$164.3 billion is a haircut of 5 1/4%. Sounds pretty generous to me, especially when 72 percent of the collateral is junk.
The cushion "was far too small for the risk of the underlying collateral," [Craig Pirrong , a finance professor at the University of Houston] said. "Collateral that's junk or defaulted debt and equities at a time when market volatility was huge is pretty eye opening."
Here's a chart from Yves Smith's book ECONNED that she put up this morning.
Those aren't the terms on which the Fed was lending money.
Here's the thing, if the Federal Reserve was so frightened about what would happen if it didn't lend freely at a low rate against junk collateral, then why aren't we making sure too big to fail banks are better capitalized? Instead we are relying on resolution authority as if that is going to stop a panic. And to top it off, people like Greenspan who Tom Hoenig says helped caused the bubble are telling us that even the lame Dodd-Frank bill with its resolution authority is too much. Are you kidding me?
So, here's the sequence of events (see The Dummy's Guide to the US Banking Crisis):
Early in the 2000s, scared of deflation, the Fed drops rates to 1%, helping to cause a housing bubble as Fed President Hoenig says.
In an anti-regulatory mood, the Fed then relaxes leverage rules for investment banks.
Investment banks leverage up as excess credit flows into housing via asset-backed securities.
The Fed hikes rates starting in 2004, aghast at the froth it has unleashed.
By Feb 2005, Alan Greenspan wonders aloud about the conundrum of easy money, animal spirits and low rates. Bernanke invents the Asian Savings glut theory as intellectual cover for the Fed's actions.
In 2006, things start to unravel when those that could least afford to purchase homes — so called subprime borrowers — start to default, prices having run well out of their range of affordability
Banks get hammered on losses starting in 2007 with the Household International credit writedown by HSBC. Fed Chair Bernanke says, "At this juncture . . . the impact on the broader economy and financial markets of the problems in the subprime markets seems likely to be contained."
Later in 2007, a liquidity crisis hits anyway, taking British bank Northern Rock down.
By January 2008, things spiral out of control, so the Fed starts lending money at low rates against collateral under the Term Auction Facility.
In March 2008, the Fed organizes a takeover of Bear Stearns by JPMorgan Chase, leading market participants to believe no one could fail.
In August 2008, Fannie and Freddie get a government backstop.
By September 2008, without a clear sign as to which firms were insolvent and which were illiquid, market mistrust creates fear.
Lehman heads to the wall with implicit thoughts of a Fed backstop due to the Bear deal. They are not bailed out. Panic ensues.
Now, the Fed acts as a lender of last resort. But it gives money to any and all takers, against whatever dodgy collateral at a low rate without significant haircuts.
Scared of deflation again , the Fed drops rates to 0%, helping to cause a commodities bubble as Fed President Hoenig says.
Does that sound like insanity to you? Question: if 1% rates for an extended period last decade got us a housing bubble, why should we expect zero rates to be a good thing? The definition of insanity is is repeating the same behaviour and expecting different results.
Just sayin'.
bailoutbankscrisisfinancial historymonetary policyregulationregulatory capture
Bill Gross on fiscal profligacy and dumping the negative real yields of treasuries
Debt Problem Grave, But Solution Elusive
The US economy is now on recession watch
Hippie-punching MMT
DavidLazarusUK says 8 years ago
Considering the quality of the assets that the Fed took as collateral at various stages of the crisis it would not surprise me that the Federal Reserve is itself technically insolvent as a consequence of its toxic assets.
Anonymous says 8 years ago
The Japanification of Europe

redpajama_set_name: RedPajamaCommonCrawl


Everyone has experienced IVRs (interactive voice response). It sounds something like this: "press 1 if you're a developer, press 2 if you're an awesome developer". Here, we will show you how to build an IVR system that use's HipChat APIs and post details of incoming calls, recordings and voicemails on your Plivo number to HipChat. After each call is complete, our bot also sends the URL of the call recording to the Chat room so that our sales team can have access anywhere anytime.
Call Forwarding - Forward the incoming call to the first point of contact (Mobile, PSTN, Browser phone, or any SIP Phone).
This app is essentially simliar to Google Voice. Think about Google Voice as an app that uses a single phone number that does routing/fowarding, transcription, email, SMS, IVR, and voicemail, which is exactly what we're building here. This means that with a few changes to this HipChat API integration, you can build your own app that essentially emulates Google Voice. Be sure to look out for our follow up post that will feature this hack and include a GUI as well.
We are going to build our app using Flask, Heroku and Plivo. The goal of this tutorial is to get you started with Plivo APIs and XML and quickly prototype and deploy apps on heroku. We will be leveraging three Plivo XML elements, GetDigits, Dial and Speak to create this application.
Get a Free Plivo Trial Account: upon signup and verification, you will receive a SMS enabled Plivo Number, your Plivo Auth ID, and your Plivo Auth Token.
Get a HipChat account: head over to the Heroku documentation to get accquianted on how to setup an account and install the Heroku toolbelt.
Install Python and Virtualenv: see this guide for guidance.
Get the latest source code by typing the following in the terminal and hit Enter.
Open the file creds.py in this folder in your favorite text editor and add the Plivo and HipChat credentials.
Get the Plivo Auth ID and Auth Token from the dashboard.
Open the file config.py in this folder in your favorite text editor. Create a HipChat roomand add it to the VOICEMAIL_HIPCHAT_ROOMS list.
Deploy your code to heroku and add the redistogo add on.
Note: For each application, Heroku provides 750 free dyno-hours. To run more than 1 dyno, you may have to verify your heroku account. Make sure you scale down the dynos when you are done testing.
We'll create a Plivo application and point it to the number you purchased in step 1. Open https://console.plivo.com/app/ in your browser.
Click on the New Application button to create an application. Give a name to your application, lets call it 'Sales Line IVR'. Click on 'Create' upon done. Use the answer URL as http://(heroku app url)/response/forward/. Choose the GET method. Leave the other fields as it is for now. Note: Replace "heroku app url" with the the url returned when creating the heroku app in Step 6.
Assign your Plivo number to your new Sales Line IVR App Choose any of your Plivo phone numbers found on your Numbers tab in the Plivo Dashboard to PLIVO_NUMBER. If you don't yet have a number, you can claim your free local US phone number in the Numbers tab when you click on the "Claim your free local US number" link.
You are all set! Make a call to your Plivo Number and test it out.
Is anything on this page unclear? Suggest edits and help us improve our documentation!


text:
Гербът на Разлог е един от основните символи на града и общината. е Утвърден е в 1987 година. Негови автори са Тодор Варджиев и Асен Ботев. Гербът е пробив в употребата на хералдични символи. Тривърхата планина символизира трите планини около Разлог – Пирин, Рила и Родопите. Орелът е златен на червен фон, тъй като и женските, и мъжките носии в Разлог са алени със сърма отгоре.

Вижте също
 Герб на Гоце Делчев

Бележки

Разлог
Разлог
Култура в Разлог
Община Разлог

redpajama_set_name: RedPajamaWikipedia


text:
Q: Word for a sentence structured like: 'Something is to something as something is to something' For example: 'Blue is to colours as five is to numbers'
You get these kinds of sentences in aptitude tests where they will typically omit the last word for you to work out. Is there a word for sentences structured like this?

A: No matter how they're written, they're called analogies:

An analogy is a statement that suggests two terms are related to each other in the same way that two other terms are related to each other. The [Miller Analogies Test] analogy items are written as equations in the form "A : B :: C : D." This can be read as either "A is related to B in the same way that C is related to D" or as "A is related to C in the same way as B is related to D."
The Miller Analogies Test Study Guide

What Were SAT Analogy Questions?
These were the original stereotypical SAT questions. You were given a pair of words and asked to choose from five other pairs of words to find the relationship that most closely resembled that of the first pair.
In the following example, you can interpret the dots as the words "is to" so you would think of it as "Paltry is to significance as _______ is to _________." where the blanks are the two words in one of the answer choices.

PALTRY : SIGNIFICANCE ::
A. redundant : discussion
    B.  austere : landscape
    C. opulent : wealth
    D. oblique : familiarity
    E. banal : originality
(the correct answer is E)

SAT Analogies and Comparisons: Why Were They Removed, and What Replaced Them?

This weekend, co-vocabularists are invited to submit novel analogies, using the tried and trusted formula: A is to B as C is to D.
Weekend Competition — NYT Opinion Pages: Schott's Vocab

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Just an hour outside of Barcelona and on the outskirts of Salou, this theme park is a great location for any thrill seeking family, for a full holiday or just a long weekend away. There are so many rides and attractions waiting for you at the other end you will never hear the dreaded half term B word (bored). This theme park can only be described by us as EPIC but then, we do love a good roller-coaster and these are pretty wild. Plus with a great range of hotels on site and off, with various themes and amenities, you'd be really pushed to get closer to the action or a better theme park weekend in Europe (well outside of the uk obviously, see my reviews of Thorpe park here).
With just a ninety minute flight there and an extra thirty minutes on return, this is actually quicker to get to than driving to other destinations in the UK. Ok, ok, so that's not adding in airport times and transfers but you get my point, if theme parks are your families thing then this is the short break you're looking for.
The Hotel Gold River.... yeeeeeeehahhhhhh!!
After a little mix up with our hotels, a break down from Kyd, a tour of Hotel PortAventura, then checking into Hotel ElPaso and finally making it to our original hotel, the beautiful Hotel Gold River, which is where we stayed for the 2 nights, we were finally there. (Mix up promptly sorted after speaking to the correct person and apparently is due to it not being booked by the routine booking process, out of our hands and there's, so don't hold it against them).
This hotel is probably one of the coolest places we've stayed at. Themed on the Wild West with cowboys throughout, you get taken back in time to a place you have only seen in the movies. With ** separate buildings each themed to a different type of old fashioned, cowboy style movie store and centred around an elegantly decorated Town Hall building, full of homely western style rooms, you couldn't get a more exciting atmosphere to start and end your day at a theme park. The best part is it is actually attached to the park with its own entrance, making life pretty easy for us families with medical needs.
What can I say about the actual park... it is very well thought out and has everything a family may wish on a day out/holiday. There are rides for everyone, with an area specifically designed for the younger members of the family, which does get pretty busy, so be aware of that. The thrill rides are exactly how they are described on the tin... thrilling. They also hold something for everyone, from slow and spiny to tall and petrifying, whether you like rides or not, there is something for you. We never got to a show as Rocky isn't the easiest toddler to get seated for shows in general but I have heard amazing things and if we go back I am definitely getting involved in that side of the park too. You are never too far away from a toilet either, making life really easy for Kyd and his medical needs. The facilities were clean and safe and I was never too aware of any escapism issues for either Kyd or Rocky. It can get very busy, I do suggest investing in an Express Pass for the main rides, even at the end of the season, October, the queues were 60mins long on the larger rides, but 12mins on the smaller, so swings and roundabouts with that.
Kyd, having a rest... it's tiring being brave you know!
This weekend wasn't without it's special needs mishaps, we had issues from the beginning but the staff were more than helpful to accommodate Kyd's needs. People with speech issues may wish to have a carer with them at all times, the Spanish staff find it hard to understand most clear English so those with speech issues can get completely lost in translation. The staff were all pretty good, but I did find that a lot of the other guests stared a lot, different culture it seems. The good news though, I saw several school trips of Special Needs children and teachers throughout the day, meaning this is a tried and tested park for such needs and that showed throughout the parks staff.
Ride Access Passes - You can get free express passes/exit passes for the rides by taking proof of disability to customer services on arrival. Your DLA letter or letter from your GP (must be stamped and dated).
Accessibility - The park is quite large and very spread out, so could be difficult for those who have difficulties walking. However, there are plenty of places to sit and relax, so don't let that put you off.
Special dietary needs - I suggest you learn the Spanish for does this contain Nuts/Dairy/whatever you aren't allowed... the serving staff don't often understand the English version of it. If you aren't sure... don't risk it.
Accommodation - Staying on site is definitely advisable. With unrestricted access to the park with your room key, it makes life easier if you need to make quick trips for medication, clothes changes, naps and so on. On that note, if you are staying at the park may I suggest picking the Hotel Gold River rather than the Hotel PortAventura, after seeing them both, the Hotel Gold River Town Hall, is more accessible for accessibility issues, less steps. Avoid the ElPaso, the distance is further to the park and although there is a fun little train, which you'd need to fold your chair up for, it makes emergency trips to your room a nightmare and take much longer out of the fun part of your day.


One year later, I noticed that most of the problems I faced here are either no longer problems, or just gone. Also, I happened to notice how wonderful the site layout is now (a blue theme is definitely better than a red theme for the new SJ)! Seeing all the new changes to the web site that caused me problems, I couldn't help but give this place a second chance!
Now I'm going to start almost from scratch and hopefully receive more benefits instead of add more problems!
AYM FanComic by SMF - Cliche Manners!
Is it just me, or do the text boxes look different?
It's fitting that GEEBS castle is ripped by a certain psychopath of a Koopa.
So, I guess that's the last we see of him, huh?
Well! It's actually a pleasant surprise to see E-Man making a cameo here! Nice reference to by old signature!
You do realize that if an apocalypse happens, you'll perish along with everything else, right?
Well, I hope you're not disappointed by the lack of an apocalypse.
You mean that one that was on Saturday? If that's so, then why am I still typing?
Wow... That owl has to be super annoying to make Link jump off a mountain that is almost impossible to climb back up again.
Well, since Wart was the final boss in SMB2, you could be onto something.
Wow... Talk about confusing, G.B.A... Well, by the next comic, I think things will be in order!
This is starting to get interesting, G.B.A! Now we have two antagonists to deal with!
Oh! You have a Deviant ART account?
Huh! Ganon is looking rather chipper today in your avatar, Lord Enigma!
What an exciting antagonist you created there, G.B.A!
Well, that's more graphical than the original battle!
All you do is just make the Saturday morning cartoon heroes look serious... But that's why we love you!
Whoa... I didn't see that coming! I had a hunch that the Mimi G.B.A was with isn't real, but the fact that she isn't real to begin with and G.B.A's old girlfriend is actually a witch caught me off guard! ........ And to think that I was the master of plot twists!
Boo's Mansion, huh? Well, I'm not surprised that BM is the cause of one of your problems, G.B.A. That place has been going downhill, but that's another story.
By the way, since Mimi is here again (or possibly some Mimi doubleganger), are you going to make her fat again?


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This page was printed on 4/20/2019 1:35:55 AM.


Wedding photographers succeed because of one reason only; they see the aspect of being a wedding photographer as more than just a job that keeps things afloat, they see it as a chance to chase after their passion. If you look at your wedding photography business as more than just a way to get money, then you are off to a pretty good start. Even if you have all of the best business plans and timelines down pat, or even the photography skills to boost; if it is not something that you are particularly passionate about, you will never be able to bring it through towards the whole thing. Passion is the difference between a well executed business plan and a business plan that will be able to make the entire thing thrive and flourish when it all comes down to it. If you are not particularly passionate about it, then you might as well forget it because you will end up wasting money in the process. Wake up to inspiration and to excitement for wedding photography every single day and you will never go wrong.
Wedding photographers go through a lot of challenges and roadblocks when they are just starting out with their careers and that is something that you should be prepping for as well. It will not be an easy ride for you at the beginning. It is always an uphill climb and this is something that you should prepare for one way or the other. Do not let these failures at the start sway you. Stand your ground as much as possible and work your way through it. The no's that you get should only inspire you all the more. Success will only be sweeter if it is something that you get to work hard for. You will learn a lot of things from the first few mistakes that you make so hang in there and stay strong throughout your climb towards the top.
Capitalize on the things that make you unique compared to the other wedding photographers out there.
Do not rush off to mimic the next big wedding photographer Birmingham who breaks out into the scene just yet. These people were able to make good on what they are doing because they banked on what they believe makes them different from the rest of the competition. You should be wise enough to make sure that you are able to do the same. The things that make you stand out are the things that will make your marketable as a wedding photographer so work on it and develop it as much as possible.
Get a job on the side just to make things easy for you as you are trying to kick your career off the ground.
You need a steady source of income while you are trying to grow your business so make sure that you at least keep some kind of job on the side while you are more or less waiting out your next big break.


Abstract: Cellulases have wide applications and biotechnological potentials for various industries. A bacterium producing an extracellular, thermostable cellulase was isolated from plant leaf litters of Lagerstroemia indica Linn, inside a botanical garden. According to morphological, biochemical and physiological characterization, it was tentatively identified as Enterobacter sp. Molecular characterization, using the 16S rRNA gene sequencing was used to confirm the identity of the bacterium as Enterobacter cloacae IP8. Effects of some cultural factors such as carbon and nitrogen sources, pH and temperature, on cellulase production from the bacterium, were investigated. Some physicochemical properties of the crude cellulase from E. cloacae IP8 were determined to evaluate its potentials for industrial applications. The maximum yield of cellulase (10.78 U/mL) was at 28 h of incubation using carboxymethyl cellulose (CMC) (1.5%, w/v), peptone (2.0%, w/v), inoculum size (1.0%, v/v), pH and temperature of 7.0 and 45 oC, respectively, and agitation speed 150 rpm. The crude cellulase exhibited optimum activity at 60 oC, retaining 75.0% of its maximal activity at 70 oC. It had optimum pH of 7.0, retaining 58.0% of its original activity at acidic pH 5.0. Metal ions Na+, Ca2+ and Mg2+ remarkably enhanced activity of the cellulase while K+ and EDTA inhibited activity of the cellulase from E. cloacae IP8. The characteristics of the cellulase from E. cloacae IP8 revealed the enzyme as being thermostable and an acidic to neutral metalloenzyme. Therefore, the enzyme from this strain could be applied in industrial applications such as lignocellulosic biomass conversion into fuel and other value added products.
Cellulases have wide applications and biotechnological potentials for various industries. A bacterium producing an extracellular, thermostable cellulase was isolated from plant leaf litters of Lagerstroemia indica Linn, inside a botanical garden. According to morphological, biochemical and physiological characterization, it was tentatively identified as Enterobacter sp. Molecular characterization, using the 16S rRNA gene sequencing was used to confirm the identity of the bacterium as Enterobacter cloacae IP8. Effects of some cultural factors such as carbon and nitrogen sources, pH and temperature, on cellulase production from the bacterium, were investigated. Some physicochemical properties of the crude cellulase from E. cloacae IP8 were determined to evaluate its potentials for industrial applications. The maximum yield of cellulase (10.78 U/mL) was at 28 h of incubation using carboxymethyl cellulose (CMC) (1.5%, w/v), peptone (2.0%, w/v), inoculum size (1.0%, v/v), pH and temperature of 7.0 and 45 oC, respectively, and agitation speed 150 rpm. The crude cellulase exhibited optimum activity at 60 oC, retaining 75.0% of its maximal activity at 70 oC. It had optimum pH of 7.0, retaining 58.0% of its original activity at acidic pH 5.0. Metal ions Na+, Ca2+ and Mg2+ remarkably enhanced activity of the cellulase while K+ and EDTA inhibited activity of the cellulase from E. cloacae IP8. The characteristics of the cellulase from E. cloacae IP8 revealed the enzyme as being thermostable and an acidic to neutral metalloenzyme. Therefore, the enzyme from this strain could be applied in industrial applications such as lignocellulosic biomass conversion into fuel and other value added products.
Cellulose is the most abundant biopolymer in nature and forms the major component of plant biomass (Yin et al., 2010; Sadhu et al., 2013; Kilo et al., 2016). It is a linear polysaccharide polymer of β-1,4-glycosidic bonds and its crystalline structure is stabilized by intramolecular and intermolecular hydrogen bonds (Hikaru et al., 2008). Globally, plants are estimated to produce 4 x 109 tons of cellulose annually (Narasimha et al., 2006; Yin et al., 2010). Most of the cellulose exist as wastes which may be agricultural, urban or industrial in origin and includes straw, corn cobs, wood wastes, peat, bagasse and waste paper (Reddy et al., 2015). These cellulosic wastes exist abundantly as organic wastes which tend to accumulate in the environment. However, they could be utilized more efficiently by being converted to fermentable sugars which act as carbon sources for the production of various products of commercial interests such as fuels and chemicals (Chen et al., 2007; Kuhad et al., 2011). The natural degradation of cellulose is carried out by cellulolytic microorganisms, which form an important part of the biosphere carbon cycle. Cellulolysis, which is the complete hydrolysis of the cellulose polymer to glucose is carried out by the synergistic action of three cellulolytic enzyme system (Acosta-Rodriguez et al., 2005; Eveleigh et al., 2005; Kuhad et al., 2011). β-1,4-endoglucanase (EC 3.2.1.4) randomly attacks the internal β-1,4-glycosidic bonds along a cellulose chain, resulting in glucan chains of different lengths; the β-1,4-exoglucanase (EC 3.2.1.91) acts on the ends of the cellulose chain and releases β-cellobiose as the end products while the β-1,4-glucosidase (EC 3.2.1.21) specifically hydrolyzes β-cellobiose to two glucose molecules. Biological oxidation of cellulose by cellulases is preferred for industrial purposes due to high yields of desired hydrolytic products with minimal by-products (Parry et al., 2001). Effective utilization of cellulosic materials through bioprocesses will be an important key to overcome the shortage of foods, feed, and fuels which the world may face in the near future, because of the explosive increase in human population (Ohmiya et al., 1997). Cellulases have wide applications and biotechnological potentials for various industries including chemical, fuel, food, brewery, animal feed, textile and pulp and paper industries (Bhat, 2000; Gilna and Khaleel, 2011; Karnouri et al., 2014; Ladeira et al., 2015). Some industrial processes for which cellulases find useful applications are clarification and liquefaction of fruit and vegetable juices, biopolishing of cotton and other cellulosic fabrics, improvement of nutritional and digestibility of animal feeds and ethanol fuel production (De Carvalho et al., 2008; Gaur and Tiwari, 2015; Behera et al., 2017). Enzyme production is closely controlled in microorganisms and therefore these controls can be carefully exploited to enhance enzyme productivity. Cellulase yield depends on a complex relationship involving a variety of factors like pH, temperature, incubation period, carbon and nitrogen sources (Immanuel et al., 2006). To establish a successful fermentation process, microorganisms are exploited for the overproduction of the desired metabolite. An elaborate investigation is thus necessary to establish the optimum conditions necessary for scale up of of enzyme production in an individual fermentation process. Studies have shown a direct proportion between production costs of cellulase and productivity of enzyme producing microbial strains (Omojasola et al., 2008). Such processes would assist in food and animal feed processing, solve modern waste disposal problem and reduce drastically man's dependence on fossil fuels by providing a convenient and renewable source of energy in the form of glucose which can be used for production of ethanol, organic acids and other commercially useful chemicals (Makoi and Ndakidemi, 2008). The conversion of cellulosic biomass by microorganisms is thus a potentially sustainable approach to developing novel bioprocesses and products (Kuhad et al., 2011). Cellulase research has been concentrated mostly on fungi but there is increase in cellulase production by bacteria due to their higher growth rate, thermostability and alkaline stablility properties (Nagendran et al., 2009; Acharya and Chaudhuri, 2012). Cellulose-degrading bacteria have been isolated from variety of sources such as soils, decayed plant materials, hot springs, organic matter, faeces of ruminants and composts and the search for bacteria with improved cellulolytic activities has been ongoing (Ray et al., 2007; Doi, 2008). There are reports of production of thermostable cellulases from several bacteria strains isolated from diverse sources. These include Bacillus vallismortis RG-07 from soil (Gaur and Tiwari, 2015), Bacillus licheniformis Vic. From geothermal wells (Kilo et al., 2016), B. licheniformis 380 from compost (De Marco et al., 2017) and B. licheniformis C55 from solid waste carrageenan (Listyaningrum et al., 2018). The objective of our study was the isolation and screening of cellulolytic bacteria from plant leaf litters of Lagerstroemia indica Linn. Optimization of different nutritional and environmental parameters for maximum cellulase production from selected bacterium and characterization of produced thermostable cellulase were then carried out.
Bacterial strains were isolated from dumpsites of plant leaf litters of Lagerstroemia indica Linn by using serial dilutions and pour plate technique. Aliquot (100 µL) of each diluted sample was spread on correspondingly labelled sterile nutrient agar plates which were incubated at 45 oC for 48 h (Apun et al., 2000). Representative bacterial colonies were purified by repeated streaking on nutrient agar and then preserved at 4 oC on nutrient agar slants for further identification and screening for cellulase production.
Pure cultures of bacterial isolates were individually streaked on carboxymethyl cellulose agar (CMCA) plates consisting of yeast extract (0.2%, w/v), KH2PO4 (0.1%, w/v), MgSO4.7H2O (0.5%, w/v), NaCl (0.075%, w/v), peptone (2.0%, w/v), agar (1.5%, w/v) and high viscosity carboxymethyl cellulose (0.5%, w/v). The CMCA plates were incubated at 55 oC for 48 h. These plates were then flooded with Congo red (0.1%) for 15 min at room temperature and then destained with 1 M NaCl solution. Clear halo zone surrounding bacterial colonies was indicative of cellulose hydrolysis and measured in centimeters (Wood and Bhat, 1998). Endo-1,4-β- glucanases (Cx) producing activities of the isolates were estimated by the carboxymethyl cellulose hydrolysis capacity (HC value: ratio of diameter of clear zone and line of streak) on the cellulose congo-red agar (Lu et al., 2005). Isolates with high HC values were selected for identification and cellulase production using submerged fermentation system.
Identification of bacterial isolate with the highest cellulolytic activity was carried out using phenotypic methods involving morphological, biochemical and physiological characterizations (Harrigan and McCance, 1976). Molecular characterization was then carried out to confirm the identity of the cellulolytic bacterium. The method was based on the 16S rRNA gene nucleotide sequencing and comparison to the sequences in National Centre for Biotechnology Information (NCBI) database (Bethesda, MD, USA). The primers used in this study were f25 (5′-AGAGTTTGATCMTGGCTCAG-3′) and r1525 (5′-AAGGAGGTGWTCCARCCGCA-3′). The PCR products were analyzed by agarose gel electrophoresis. ABI 3130 x L Genetic Analyzer (Applied Biosystems, California, USA) was used for the nucleotide sequencing. Sequence similarity search of the Genbank data was done using a Basic Alignment Search Tool (BLAST) from the NCBI database (Mangamuri et al., 2012).
Cellulase was produced by submerged fermentation (SmF) in Erlenmeyer flasks (250 mL) containing 100 mL of basal medium composed of yeast extract (0.2%, w/v), KH2PO4 (0.1%, w/v), MgSO4.7H2O (0.5%, w/v), NaCl (0.075%, w/v), peptone (2.0%, w/v) and high viscosity carboxymethyl cellulose (0.2%, w/v). The medium was inoculated with standardized aqueous suspension of the bacterium from a 24 h old culture (3.0 x 106 CFU/mL). The mixture was incubated at 45 oC for 48 h on a rotary shaker at 150 rpm. After incubation, the fermentation broth was centrifuged at 6000 rpm for 30 min at 4 oC to obtain the cell-free supernatant (CFS) that was collected as the crude enzyme extract and used for cellulase assay.
The β-1,4-endoglucanase activity towards carboxymethyl cellulose (CMC) was measured by the appearance of reducing end groups released by the action of the enzymes on the substrate using the method of Somogyi (1952). Mixture of 0.1 mL of 0.1%, w/v CMC (in 100 mM of phosphate buffer, pH 7) with 0.5 mL of crude enzyme was prepared. Phosphate buffer (0.85 mL) was added to make the corresponding volume 1.0 mL and the resulting mixture incubated at 55 oC for 20 min. The reaction was terminated by the addition of 1.0 mL combined copper reagent. The reaction mixture was heated at 100 oC for 20 min in a boiling water bath. The tubes were cooled rapidly under running tap water and arsenomolybdate reagent (1.0 mL) was added to all the tubes while shaking. The mixtures were diluted with distilled water (7.0 mL). The optical density (O.D) was read at 540 nm against blank. The amount of reducing sugars produced was interpolated from the glucose standard curve. One unit of cellulase activity was expressed as the amount of enzyme that liberated reducing sugar equivalent to 1 µg of glucose per milliliter per minute under the specified assay condition.
The time course of cellulase production was determined and compared with bacterial growth according to the method described by de Souza and Martins (2001), with appropriate modifications. The growth curve and enzyme production for the bacterium was determined by inoculating a 250 mL enzyme fermentation medium with 2.5 mL standardized inoculum of bacterium in an Erlemenyer flask. The culture medium was incubated at 45 oC for 48 h with agitation at 150 rpm. At 2 h intervals, 5.0 mL samples were collected aseptically for a period of 48 h. The optical density of the culture was checked at 680 nm using spectrum lab 23A spectrophotometer and recorded as the cell optical density. This was then centrifuged at 6000 rpm for 30 min to obtain cell-free supernatant (CFS). The cellulase activity of the clear supernatant was then measured.
Fermentation medium (50.0 mL) adjusted to different pH conditions 4.0 to 10.0 were each inoculated with 0.5 mL of standardized bacterial inoculum and the culture incubated at 45 oC for 28 h with agitation at 150 rpm. After incubation, cells were removed by centrifugation and the amount of cellulase present in supernatant was quantified.
Influence of temperature on cellulase production was determined by varying the incubation temperature from 35 to 60 oC, at pH 7.0 and agitation speed of 150 rpm. After incubation, cells were removed by centrifugation and the amount of cellulase present in supernatant was quantified.
The effect of various carbon sources on cellulase production from bacterium was studied by replacing high viscosity carboxylmethyl cellulose (0.2%, w/v) with different sugars: lactose, soluble starch, glucose, sucrose, galactose, and mannitol. The fermentation medium, previously adjusted to pH 7.0, was inoculated with 0.5 mL of the cell suspension and the culture incubated for 28 h at 45 ºC with agitation at 150 rpm. The supernatant obtained was then tested for cellulase activity.
The effect of different concentrations of carboxymethyl cellulose (0.5, 1.0, 1.5, 2.0 and 2.5%, w/v) on cellulase production from bacterium was studied. The culture was incubated for 28 h at 45 ºC with agitation at 150 rpm. The supernatant was obtained and cellulase production was determined.
The effect of different nitrogen sources peptone, ammonium sulphate, potassium nitrate, ammonium chloride and yeast extract at 2.0%, w/v, separately, on cellulase production from bacterium was studied. The fermentation medium was incubated for 28 h at 45 ºC with agitation at 150 rpm. After incubation, the supernatant obtained was tested for cellulase activity.
The effect of different pH conditions on activity of crude cellulase was determined by incubating the crude enzyme extract with 0.1 mL of 0.1%, w/v CMC prepared in different buffers (pH 4.0 to 9.0). The cellulase activity was determined according to standard procedure. The pH was adjusted using 10 mM sodium acetate buffer (pH 4.0 to 5.5) and 100 mM phosphate buffer (6.0 to 9.0). Incubation was at 45 oC for 20 min.
The effect of temperature on activity of crude cellulase was determined by incubating the reaction mixtures at temperature conditions ranging from 35 oC to 80 oC for 20 min. Thereafter, the cellulase activity was determined according to the standard procedure earlier described.
The effect of metal ions (K+, Na+, Mg2+ and Ca2+), at different concentrations (0 to 200 mM, on cellulase activity was determined by adding each metallic ion to the reaction mixture. Effect of ethylenediamine tetraacetic acid (EDTA) on activity of cellulase was determined by incubating different concentrations of EDTA (0, 4, 12, 15, 20, 30, 50, 80, 100, 120 mM) with the reaction mixture. Cellulase activity was determined in each case by following standard assay procedure.
All data were subjected to statistical analysis for determination of mean and standard deviation using SPSS version 16. Experiments were carried out in triplicates.
In this study, seven bacterial isolates were obtained from decayed plant leaf litters of Lagerstroemia indica Linn and subjected to screening for cellulolytic activity using the plate method. Among these seven bacterial strains, three were found to exhibit cellulolytic activities out of which the isolate with the highest carboxymethyl cellulose hydrolytic capacity was selected for further studies (Tab 1). Cellulolytic bacteria have been isolated from diverse environments by previous researchers. Gao et al. (2011) reported a cellulolytic bacterium, Vibrio xiamenensis, isolated from mangrove soil in Xiamen, Fujian province of China. Bacterial species showing endo- and exoglucanase activities in the sediment from a mangrove located in Ilha do Cardoso, SP, Brazil were reported by Soares Junior et al. (2013). Also, the bacterium Bacillus sp. C1, isolated from cow dung, was found to possess good potential for endoglucanase production (Sadhu et al., 2013) while the cellulase-producing bacterium Micrococcus sp. SAMRC-UFH3 was obtained from saw dust/wood shavings by Mmango-Kaseke et al. (2016). Cellulose forms the major component of plant cell wall along with hemicellulose and lignin and these complex polymeric compounds together form the bulk of the organic carbon in soil. Microorganisms are responsible for the recycling of this organic carbon in nature (Wang et al., 2008). Researchers have continuously searched for new microorganisms with higher cellulase production potential and hence important biotechnological applications (Ray et al., 2007).
Cellulolytic bacterium was identified by using morphological, biochemical and physiological characterizations (Tab 2). The cells were Gram negative, rod-shaped, non-spore forming and motile. It is an aerobic and catalase producing strain. It tested positive for gelatin hydrolysis, aesculin hydrolysis and could utilize a wide array of carbohydrates, including maltose, lactose, mannitol, raffinose and arabinose as sole source of carbon. From these various tests, isolate was tentatively identified as Enterobacter sp. Molecular characterization, based on 16S rRNA nucleotide sequencing and comparison to the sequences in NCBI database, was then carried out to confirm the identity of the bacterium as Enterobacter cloacae IP8 with maximum identity of 94% to other Enterobacter cloacae (Accession No. NR 118568.1).
The production of enzymes such as cellulase is often dependent on growth of the bacterium in the appropriate media. Cellulase production was evident in the culture supernatant after 8 h of incubation with the lag phase of 6 h during the incubation period. Enterobacter cloacae IP8 had a lag phase of about 6 h. The peak of cellulase production was at 28 h. The production of cellulase increased rapidly with increase in bacteria cell growth and peaked at the exponential phase followed by a decrease in cellulase production as the cell growth aged into the stationary phase. Results are shown in Figure 1. These results are similar to those reported by Prakash et al. (2008).
Table 2 Morphological, biochemical and physiological characteristics of E. cloacae IP8.
Optimum pH for cellulase synthesis from Enterobacter cloacae IP8 was 7.0 with enzyme activity 5.45 ± 0.55 U/mL (Figure 2). A similar optimum pH of 7.0 was observed for cellulase production from Paenibacillus sp. isolated from molasses (Islam and Roy, 2018). However, optima pH of 5.0 was reported for cellulase production from E. cloacae WPL 214 isolated from bovine rumen fluid waste (Lokapirnasari et al., 2015). Cellulase activity was highest (3.02 ± 0.435 U/mL) at temperature of 45 oC (Figure 3). Similar optimum temperature value was reported for cellulase secretion by a strain of E. cloacae (Lokapirnasari et al., 2015). However, optimum temperature for cellulase production from a strain of Paenibacillus sp. was 45 oC (Islam and Roy, 2018). Temperature is a vital environmental factor which controls the growth and production of metabolites by microorganisms and this usually vary from one organism to another (Kumar and Takagi, 1999). During enzyme production at differing temperatures, stabilization of metabolic network is supposedly by the folding of proteins of the metabolic network itself, most probably by acquiring chaperones (Borges and Ramos, 2005).
Figure 1 Growth and cellulase production of Enterobacter cloacae IP8 at 45 oC and pH 7.0.
Maximum cellulase yield was achieved with the use of CMC (4.38 ± 0.08 U/mL) as carbon source followed by glucose (2.78 ± 0.02 U/mL) (Figure 4). Therefore, the cellulase produced can be described as an inducible enzyme, whose production depends on the presence of the substrate which also acts as its inducer. Das et al. (2010) and Islam and Roy (2018) observed that CMC was the best carbon source for cellulase synthesis by thermophilic Bacillus species isolated from cow dung and Paenibacillus sp. isolated from molasses, respectively. The best CMC concentration for enzyme production was 1.5%, w/v (Figure 5). Cellulase production was most pronounced with the use of peptone (3.51 ± 0.38 U/mL) as the nitrogen source in the fermentation medium (Figure 6). Peptone was also found to be the best nitrogen source for maximum cellulase production from Bacillus sp. Y3 (Lugani et al., 2015) and Bacillus sp. K1 (Paudel and Qin, 2015). The presence of external nitrogen source is essential in the fermentation medium during extracellular enzyme production for effective utilization of soluble carbohydrates (Ariffin et al., 2008). The use of organic nitrogen sources as compared to inorganic sources was found to be more suitable for maximum cellulase production (Ray et al., 2007).
The activity of crude cellulase from E. cloacae IP8 was determined at different pH range 4.0 to 9.0. The crude cellulase exhibited activity at broad pH range 4.0 to 9.0 with optimum activity being at the pH 7.0 (Figure 7). It retained 58.0 % of its maximal activity at acidic pH 5.0. This enzyme can therefore be classified as being active at acid to neutral pH. Similar optimum activity were also found at pH 7.0 for cellulase from Cellulomonas sp. YJ5 (Yin et al. (2010) and Bacillus vallismortis RG-07 (Gaur and Tiwari, 2015). However, optima pH of 5.0 and 9.0 were reported for cellulase from Bacillus licheniformis 380 (De Marco et al., 2017) and B. licheniformis Vic. (Kilo et al., 2016), respectively. Increasing or decreasing the pH changes the ionic state of ionizing side chains in a protein, distrupts ion pairs, breaks hydrogen bond, and consequently denatures the protein (Rawn, 1989). Cellulases are generally stable over a wide range of pH from 5.0 to 10.0 (Kim et al., 2005; Tahir et al., 2009).
The activity of crude cellulase from E. cloacae IP8 was determined at different temperatures ranging from 35 °C to 80 °C. The results showed that the enzyme had optimum activity at 60 °C (Figure 8). The enzyme retained 75% of its maximal activity at 70 °C. Optimum activity of the enzyme obtained at 60 °C indicates that the cellulase from E. cloacae IP8 was relatively thermostable. Above 70 oC, cellulase activities decreased sharply. The obtained result from the present study is similar to the findings of Yin et al. (2010) and De Marco et al., (2017) on cellulase from Cellulomonas sp. YJ5 and Bacillus licheniformis 380, respectively, which had optimum temperature of 60 ºC. However, lower optima temperatures of 35 oC and 50 oC were reported of cellulases from a strain of Pseudomonas fluorescence (Bakare et al. 2005) and Bacillus licheniformis C55 (Listyaningrum et al., 2018), respectively. Cellulase from E. cloacae IP8 thus exhibited relatively higher optimum temperature for activity and good thermal stability. These properties are very important for cellulose saccharification and may make the enzyme suitable for application to several biotechnological and industrial purposes.
The effect of various metal ions on the crude cellulase from E. cloacae IP8 was tested (Figure 9). The cations Ca2+ and Na+ exhibited stimulatory effects on the enzyme activity in all range of concentrations (Figure 9). While Mg2+ concentrations 4 mM to 15 mM inhibited the activity of the enzyme, it stimulated its activity from concentrations 20 mM to 200 mM (Figure 9). The stimulatory effect of Na+ and Ca2+ on cellulase activity had been reported by Femi-Ola and Olowe (2011) and Gaur and Tiwari (2015) which is similar to the finding of this study. The cation K+ exhibited inhibitory effect against cellulase activity accross all range of concentrations up to 200 Mm (Figure 9). This results show that cellulase from E. cloacae IP8 is metal ion dependent. EDTA at concentrations above 4 mM was inhibitory to the activity of the crude cellulase from the bacterium (Figure 9). The result of this study is in agreement with the reported inhibitory effect of EDTA on cellulase activity by Oikawa et al. (1994). EDTA as a metal chelating agent probably acts by inactivating the cellulase either by removing metal ions from the enzyme through the formation of coordinating complex or binding inside the enzyme as a ligand, as had been noted by Schmid (1997).
In this study, Enterobacter cloacae IP8 was successfully isolated from plant leaf litters of Lagerstroemia indica L. Enhanced production of cellulase by the bacterium was achieved at pH and temperature conditions 7.0 and 45 oC, respectively and with the use of carboxymethyl cellulose and peptone as carbon and nitrogen sources, respectively. The crude cellulase of this isolate exhibited its optimum activity at pH of 7.0 and temperature of 60 ˚C. The enzyme was stimulated by Ca2+, Na+ and Mg2+ while K+ reduced its activity. Because of its activity and stability at high temperatures, cellulase of Enterobacter cloacae IP8 isolated in this study has promising potentials for biotechnological and industrial applications. However, further studies on optimization of cost-effective substrate for bulk production of enzyme and protein engineering for more thermostable enzyme with even more turn over and specific activity are needed.
Acknowledgments: Authors appreciate the Departments of Microbiology and Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria for provision of chemical reagents and equipments used.
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