Prokaryotic Diversity from Extreme Environments of Pakistan and Its Potential Applications at Regional Levels

1 Prokaryotic Diversity from Extreme Environments of Pakistan and 2 its Potential Applications at Regional Levels 3 4 Raees KHAN*1,2, Ϯ, Muhammad Israr Khan3, Ϯ, Amir Zeb2, Ϯ , Nazish Roy1 Ϯ , Muhammad 5 Yasir4, Imran Khan5, Javed Iqbal Qazi6, Shabir Ahmad7, Riaz Ullah5 and Zuhaibuddin 6 Bhutto8 7 8 1Department of Applied Bioscience, Dong-A University, Busan, Republic of Korea 9 10 2Department of Biotechnology, Quaid-I-Azam University, Islamabad, Pakistan 11 12 3Department of Plant sciences, Quaid-I-Azam University, Islamabad, Pakistan 13 14 4Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, 15 Jeddah, Saudi Arabia 16 17 5Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia 18 19 6Department of Zoology, University of the Punjab, Lahore, Pakistan 20 21 7Department of Microbiology and Biotechnology, Sarhad University of Science and Information 22 Technology, Peshawar, Pakistan 23 24 8Department of Computer system Engineering, BUET Khuzdar, Pakistan. 25 26 ϮThese authors contributed equally to this work. 27 28 * Correspondence: 29 Dr. Raees Khan 30 [email protected] 31 32 33 Keywords: Extremophiles, Diversity, Pakistan, Extremozymes, Biotechnological potentials 34 35 Number of words 36 37 [Total number of words; 7129, total figures and tables; 6] 38 39 40 41 42 43 44 45

46 Abstract 47 Extremophiles, the microorganisms thriving in extreme environments, provide valuable resources 48 for practicing novel biotechnological processes. Pakistan homes a wide spectrum of extreme 49 environments which harbor various biotechnologically significant microorganisms. This review 50 gauges the structural and functional bacterial diversity of several extreme environments, 51 emphasizing their potentials as a source of extremozymes, and in bioleaching, bioremediation, 52 and bioenergy production at regional level. Further, this review highlights a panoramic account of 53 the local natural conservatories of extremophiles. The inadequacies of current fragmental 54 research are discussed with suggestions to quantitatively define the structural and functional 55 diversity of unexplored extreme localities. 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92

93 94 1. Introduction 95 Around half a century ago, people were largely unacquainted with the term extremophile, making 96 it rather difficult to comprehend that life could exist at pH as low as 1, temperatures higher than 97 100 °C, high salinity, under 1400 atm atmospheric pressure and high concentrations of various 98 recalcitrant substances [Hough and Danson, 199; Podar and Reysenbach, 2006]. However, like 99 other discoveries, this hidden mystery was unmasked with the exploration of novel microbial 100 communities – that remodeled the anthropocentric notions of habitable environments [MacElroy, 101 1974; Hendry, 2006]. Such microorganisms capable of both withstanding and surviving the 102 extreme environments were distinguished thus named extremophiles [Podar and Reysenbach, 103 2006]. Subsequently, specific terms such as acidophiles, halophiles, thermophile, barophiles and 104 psychrophiles representing the nature and corresponding habitat fascinated initially the 105 microbiological and later biotechnological literature [Hendry, 2006; Cavicchioli et al., 2011]. An 106 arsenal of specialized lipids, enzymes and proteins combat the austere constraints to fortify the 107 structural and functional attributes of the cellular machinery in extremophiles [Berezovsky and 108 Shakhnovich, 2005; Chen et al., 2005; Fukui et al., 2005; Falb et al., 2005; Choi et al., 2006]. 109 110 The unique nature and interesting physiology of extremophiles piqued the interest of researchers 111 and scientists to appraise the nature and dynamics of extremophiles and their derived components 112 that could be a great frontier for biotechnological industry [Podar and Reysenbach, 2006]. The 113 extremophile-derived components are already being applied in molecular biology, food 114 industries, cosmetics, health sector, textile industries and waste processing units [Cavicchioli et 115 al., 2011]. The need to study the abundance, activity, diversity and distribution of 116 microorganisms in extreme environments at regional level for conserving their biodiversity and 117 understanding the mechanisms of their survival in the unusual environments responding to the 118 current global climate change are indispensable on cogent grounds. Furthermore, studies at 119 regional level may strengthen our knowledge apropos of diverse nature of extremophiles 120 inhabiting the same range(s) of extreme environment(s), but at different geographical locations. 121 122 Around the world, regional efforts have been carried out to evaluate structural and 123 functional attributes of extremophiles’ diversity; for instance United States [Horikoshi, 1998], 124 United Kingdom [Norton et al., 1993], India [Ghosh et al., 2003], China [Hu et al., 2015], 125 Australia [Conner and Benison, 2013] and some African countries [Jones and Grant, 1999] are 126 actively contributing to extremophile science. In this review, we focus on the extremophilic 127 bacterial diversity and their habitats from various geographical regions of Pakistan discussing the 128 so far explored extreme environments of the country and their reported bacterial diversity. 129 Further, the review highlights possible biotechnological applications of locally isolated 130 extremophiles for biotechnological industries. Moreover, new methods have been suggested for 131 structural and functional evaluation of local virgin extreme sites. 132 133 1.2 Geographical importance of Pakistan 134 Pakistan is the 36th largest country and is located at 241o to 371o N latitudes and from 611o to 135 761o E longitudes. The total land area is about 8×105 km2, which is expanded 1700 km from 136 Northeast to Southwest with a total width of approximately 1000 km from East–West. The 137 country has wonderful geomorphology that includes the lofty mountains of Himalayas, 138 Karakorum, Hindukush, and Pamirs in the North with the fascinating coastline of the Arabian Sea 139 in the South. In the central part of the country, mountains are bounded by the fertile plains of

140 River Indus. One of the world’s largest deserts, known as Thar, is partially located in the Eastern 141 part of the country. Also, the volcanic arc placed in Chagai, vast tectonic depression of Kharan. 142 The westward swinging mountain ranges of Makran further enhance the importance of Pakistan 143 [Zaigham et al., 2009]. Pakistan holds great geostrategic location with a diverse variety of natural 144 resources that home many life forms. Various localities in the country, from which extremophiles 145 were isolated, are summarized in Fig. 1 and Fig. 2. 146 147 2.1 Thermophiles 148 Thermophiles are the organisms that dominantly inhabit hot environments [Urbieta et al., 2015]. 149 These heat-loving microorganisms are homed in geothermally hot springs, hot soil, deep sea 150 marine hydrothermal vents and sediments of volcanic islands [Mehta and Satyanarayana, 2013; 151 Ebrahimpour and Kariminik, 2015]. They are also ubiquitous in hot polluted rivers and hot 152 composts [Ebrahimpour and Kariminik, 2015]. Depending on the habitats thermophiles are 153 classified into moderate (40–70 °C), extreme (above 70 °C) and hyper–thermophiles (80 – 154 105°C) [Ghosh et al, 2003; Reysenbach et al., 2002]. Thermophilic microorganisms are of 155 immense importance in biotechnological research, as the organisms themselves and their derived 156 products are widely used in food industry, textile industry, molecular research, bioleaching and 157 pharmaceuticals [Ebrahimpour and Kariminik, 2015, Chaudhary and Qazi, 2008; Muhammad et 158 al., 2009]. 159 160 The global seismic belt passing through Pakistan results in the formation of volcanic and 161 geo-pressurized thermal zone that sprouts out in the form of geothermal hot springs. Some of 162 these hot springs are located in Murtazabad, Budelas, Tata Pani, Mashkin, Sassi and Chu Tran 163 areas [Zaigham et al., 2009]. These hot springs are of local and national interest, as they are 164 considered not only the potential source of renewable energy but also medicinally important 165 [Javed et al., 2009]. The culture-based methods employed by few studies identified that Bacillus, 166 Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Salmonella and Thermus 167 aquaticus species inhabit these springs [Javed et al., 2012; Saleem et al., 2012]. The thermophiles 168 identified through culture-based methods from various environments of Pakistan are presented in 169 Table 1. Interestingly, few industrially-beneficial strains such as Thermus aquaticus, Geobacillus 170 pallidus sp. have been identified from these sites [Javed et al., 2012; Zahoor et al., 2012]. 171 Bacillus licheniformis [Qadar et al., 2009; Niaz et al., 2010; Ghumro et al., 2011] and various 172 other species of Bacillus have been also reported [Zaidi, 2007; Rafique et al., 2010; Khan et al., 173 2011; Asad et al., 2011]. These thermophilic isolates produce industrially important enzymes, 174 including glucosidases, gelatin hydrolyses, extracellular α–amylases, cellulases, alkaline 175 proteases, endoglucanases, and cellobiohydrolases. Majority of the enzymes have been reported 176 stable at higher temperatures and salt concentrations. Additionally, some of the isolated 177 thermophiles were capable of producing ethanol using sugarcane bagasse as a carbon source 178 [Chaudhary and Qazi, 2008]. Few of the isolated thermophilic strains with antimicrobial 179 properties could be further investigated as a potent source of therapeutic compounds [Muhammad 180 et al., 2009]. Some of the isolated thermophiles were even capable to depyritize coal, oxidize iron 181 and sulphur [Munawar et al., 2007]; whereas, others exhibited potential to leach out metals, 182 (Table 1) [Ilyas et al., 2007]. 183 184 2.2 Psychrophiles 185 Contrary to thermophiles, the psychrophiles grow and reproduce at low temperature ranging from 186 10 °C to −20 °C [Hough and Danson, 1999; Podar and Reysenbach, 2006]. Many psychrophilic

187 species, belonging to both Gram–negative and Gram–positive bacteria, have been reported 188 worldwide from various habitats including soil, fresh water marine lakes, sandstone, sea ice and 189 oceans [Morita, 2000]. The psychrophilic bacterial enzymes are unique in catalytic activity and 190 stability; having low activation energy that results in highly efficient catalytic reactions at lower 191 temperatures [Podar and Reysenbach, 2006]. These bacteria may serve as excellent model 192 systems to understand the molecular basis of low-temperature adaptation [Podar and Reysenbach, 193 2006]. 194 195 Pakistan homes some of the world’s highest and most spectacular mountains. Surprisingly, 196 13 out of 30 world’s tallest peaks are located in Pakistan, including K2 (8,611 m), Nanga Parbat 197 (8,125 m) and Tirich Mir (7,690 m) in the Hindu Kush mountainous range. Because of the 198 numerous high mountains and abundant precipitation characteristic of a monsoon climate, the 199 mountains of northern areas of Pakistan, including the Hindu Kush, Hindu Raj, Kohistan ranges, 200 Nanga Parbat Massif and Karakoram Himalaya, has some of the largest and longest mid-latitude 201 glaciers on earth. The glacierized area is estimated to cover 15,000 km2, and as much as 37 202 percent of the Karakoram region is covered by glaciers [Williams and Ferrigno, 2010]. The cold 203 climate of this huge glacierized area might serve as a habitat for many cold-loving bacteria. 204 Compared to the magnitude of diversity of the Psychrophylic environments the reports of 205 bacterial diversity from such environments are scarce. However, a handful of the isolates reported 206 from Batura, Hopper and Passu glaciers belong to genera of Pseudomonas, Arthrobacter, 207 Stenotrophomonas and Bacillus [Ahmad, 2010] (Table 2). This calls for reconnaissance to help 208 bring to light the structural and functional Psychrophylic diversity of remote and neglected 209 environments of Pakistan. For this sake improved cultivation based techniques and metagenomics 210 may be employed to peruse the phylogeny and functional capabilities of these organisms. 211 212 2.3 Metallophiles 213 Metallophiles are the microbes that can withstand and tolerate environments characterized with 214 high metal(s’) concentrations [Brakstad and Bonaunet, 2006; Head et al., 2006]. Mechanisms of 215 metals’ uptake by these organisms, including biosorption and bioaccumulation processes, have 216 got attention of the scientific community since long—because of their promising applications in 217 the environment, agriculture, and industry [Tsezos, 2001]. A bracket of conceivable habitats of 218 metalophiles may encompass natural mining sites, industrial biotopes, deep hydrothermal vents, 219 volcanic areas and the nickel rich ultramafic soils [Mergeay, 2006]. Pakistan is gifted with 220 various natural mineral reservoirs that include antimony, aragonite, barite, celestite, chromium, 221 copper, gypsum, marble, salt and sodium compounds. Moreover, the largest iron deposits in the 222 country are situated in western Punjab province with varying iron contents of 32–34% [Kuo, 223 2007]. Other ores include the low-grade uranium ores of Siwalik Baghalchur sandstone deposits 224 in Suliman range [Khan, 1979]. Most of the reported metallophiles from Pakistan, however, have 225 not been isolated from natural metallophilic environments. Therefore, it is necessary to harvest 226 metallophiles from metallophilic habitats and to utilize their potentials. Metallophiles isolated 227 from various environments of Pakistan are listed in Table 3. Metallophiles have adapted various 228 approaches for coping with metals in corresponding biotopes. For instance, C. freundii, K. 229 oxytoca and B. anthracis produce arsenic reductase enzymes that successfully reduce As(V) into 230 As(III) and thus proceed to leach out arsenic [Shakoori et al., 2010]. Other metallophiles such as 231 S. thermosulfidooxidans strains have been found to solubilize not only sulfide ores but other 232 metals as well [Afrasayab et al., 2002]. Additionally a variety of metallophillic isolates such as 233 Pseudomonas and Sulfobacillus spp., [Rehman et al., 2009], Vibrionacea sp. [Afrasayab et a.,

234 2002], T. thiooxidans, T. ferrooxidans [Ilyas et al., 2007], S. thermosulfidooxidans and A. 235 ferrooxidans [Rehman et al., 2009] were able to tolerate and leach out a variety of metals 236 including uranium, chromium, arsenic, and mercury. 237 238 The biotechnological potentials of metallophiles from Pakistan can be utilized locally in a 239 variety of ways; for instance, in commercial bioleaching operations and a wide range of 240 bioremediation projects. Engineered bioleaching is already being used in many countries like 241 Australia, South Africa, Chile, Myanmar, Ghana, Brazil and Peru for mining metals such as gold, 242 cobalt and copper [Brierley and Brierley, 2001]. Employment of endogenous metallophiles for 243 bioleaching of low-grade ore heaps, such as those of iron and uranium, may pave the 244 development of biohydrometallurgy in the country. Bioremediation of various metal 245 contaminants, on the other hand, is of great interest amongst scientific communities [Xiong et al., 246 2008; Gadd, 2010; Guo et al., 2010; Monachese et al., 2012; Rajkumar et al., 2012; Basha and 247 Rajaganesh, 2014]. A large number of industries in various cities of Punjab have led to the 248 contamination of both ground as well as surface waters with pesticides, metals, and other toxic 249 agents. The factories are dispensing toxic levels of Ca2+ Cl−, Ag+, Na+, K+, Mg2+, HCO3-, arsenic, 250 mercury, iron, copper, lead, chromium, zinc, cadmium, cobalt and nickel [Azizullah et al., 2011]. 251 Improper effluent disposals and misuses of agrochemicals have further exacerbated the problem. 252 Merely a percent of these pollutants are properly treated prior to disposal [Azizullah et al., 2011]. 253 There are various reports that these pollutants are badly affecting local people’s health [Azizullah 254 et al., 2011; Arain et al., 2009; Farooqi, 2015]. Such cases call for an indispensable requisite to 255 develop new, environment-friendly methods for effluent treatments to minimize environmental 256 contamination. A potential approach to address the metal contamination is bioprocessing of the 257 effluents. Mini scale biological waste water treatment plants, aided with metallophiles—if 258 implemented in the local industries—may substantially reduce the metal load of wastewaters. 259 260 2.4 Halophiles 261 Halophiles are the extremophiles which flourish in saline habitats. This group of extremophiles is 262 further categorized into mild, moderate and extreme halophiles on the basis of salt concentration 263 of their habitats. The hyper-saline environments are diverse around the world and few of the 264 well-known natural halophilic places include Lake Magadi (Kenyan Rift Valley), Owens Lake 265 (California), Wadi Natrum Lake (Egypt) and several other saline soda lakes and soils [Oren, 266 2006; Qazi, 2013]. In addition to afore mentioned halophilic environments, coastal areas, deep- 267 sea water, and underline salt mines are potential habitats for halophiles [Oren, 2006]. 268 269 Pakistan is home to a variety of halophilic environments. Some of the local halophilic 270 biotopes include the huge Salt range and mines of Khewra, Warcha and Kalabagh [Qazi, 2013], 271 Noshpho [Khan et al., 2011], Uchhali Salt Lake [Hameed and Ashraf, 2008] and deep-sea 272 environments Halophiles and alkalophiles, isolated from various halophilic environments of 273 Pakistan are summarized in Table 4. From the Karak salt mine, different halotolerant strains 274 belonging to various genera including Oceanobacillus, Thalassobacillus, Terribacillus, 275 Brevibacterium, Halomonas, Pseudomonas and Enterobacter have been isolated utilizing culture 276 based techniques [Roohi et al., 2012]. Few of the locally isolated halophilic strains have been 277 analyzed to produce biotechnologically potent enzymes (Table 4). A halophilic Bacillus strain, 278 isolated from Khewra salt range produced thermostable extracellular alkaline protease that was 279 functional at 80 °C [Sehar and Hameed, 2011]. Other halophilic isolates from Khewra salt mine 280 including B. lichniformis, B. subtilis, K. oxytoca, B. megatarium, B. polymyxa, B. pumilis, B.

281 brevis, B. macerans and B. coagulans produced thermostable proteases which remained active 282 over a broad range of alkaline pH [Akhtar et al., 2008]. Despite the interesting functional 283 capabilities of these organisms and the diversity of halophilic extremophiles from Pakistani 284 environments there still persists a dearth of reports about such isolates. To the best of our 285 knowledge, not even a single report on the halophilic diversity of salt lakes and deep sea saline 286 environment of the country has yet been reported which leaves a huge chasm for determining the 287 structural and functional diversity of halophilic bacteria from these niches with robust 288 culturomics and deep sequencing methodologies. 289 290 2.5 Acidophiles 291 Acidophiles are the organisms that can grow and survive in acidic habitats [Canganella and 292 Wiegel, 2011]. Such environments are particularly remarkable due to low pH (<5) causative of 293 the microbial activity rather than the habitat itself [Gonzalez-Toril et al., 2013]. The Tinto River 294 in southwestern Spain, a 100-km-long river; it has a low pH (between 1.5 and 3.1) and high 295 concentrations of heavy metals in solution (iron at 0.4 to 20.2 g/L, copper at 0.02 to 0.70 g/L, and 296 zinc at 0.02 to 0.56 g/L) is one such example of an extreme acidic ecosystem [Gonzalez-Toril et 297 al., 2013]. The origin of acidic habitats has been considered to be associated with mining of 298 metals and coal [Johnson, 1998]. The acidophiles usually thrive in natural or manmade metal rich 299 habitats, mentioned afore in the metallophiles section. A variety of acidophilic bacterial isolates 300 has been reported from several environments of Pakistan. Table 2 represents a summary of such 301 micro-organisms. Among these acidophilic isolates, various Lactobacillus strains showed not 302 only antagonistic activities against many pathogens but they may also be used in the production 303 of good quality yogurt. Fermentation of animal feeds, acid tolerance, yogurt fermentation and 304 antimicrobial characteristics of the bacterial isolates render them possible candidates for their use 305 as probiotics [Aslam and Qazi, 2010]. Similarly, other acidophilic strains such as L. brevis, L. 306 plantarum, L. fermentum, L. paracasei [Ikramulhaq and Muktar, 2006, Saeed et al., 2009], S. 307 thermophiles and L. bulgaricus [Sameen et al., 2010] represent a potential source of starter 308 cultures for different biological processes, such as production of fermented bakery products and 309 good quality cheese with enhanced shelf life. Additionally, S. thermophiles and L. bulgaricus 310 strains when compared with commercially available cultures for the production of mozzarella 311 cheese showed faster acid production and efficient proteolysis which resulted in cheese with 312 higher moisture content [Sameen et al., 2010]. 313 314 The acidophilic extremophiles from Pakistani environments have a substantial economic 315 potential to be utilized for production of fermented dairy products in the well-established dairy 316 industry. Pakistan produces an estimated 42 billion liters of milk per annum [Shahid et al., 2012] 317 and is the 5th largest producer of milk in the world. Though the industrial volume of dairy 318 products could reach US$26 billion yet total milk production does not even suffice the domestic 319 human needs, amounting to various problems [Shahid et al., 2012]. Utilizing the potentials of 320 extremophilic microorganisms, especially those with probiotic characteristics and low or neutral 321 pH stable enzymes in dairy fermentation processes, may improve the quality and availability of 322 cost-effective fermented milk products, including cheese, yogurt and various other dairy 323 products. 324 325 Some of the local acidophilic isolates also possessed bioleaching capabilities. For instance strains 326 of T. thiooxidans, T. ferrooxidans and Azotobacter are capable of leaching out a variety of metals. 327 Additionally, these strains have been found to play an important role in metals

328 hyperaccumulation, nitrogen fixation and metal sulfide ores’ solubilization in their natural 329 environments, such as in waste water and in soil (landfills) [Mehmood et al., 2009]. Another 330 study showed that acidophilic heterotrophs such as S. thermosulfidooxidans were even capable of 331 recovering metals from electronic scrap with leachabilities reaching up to 80% for Ni, Cu, Al, 332 and Zn [Ilyas et al., 2007]. Moreover, coal biodepyritization studies conducted with pure and 333 mixed consortia of some of the local acidophilic isolates revealed higher biodepyritization rates 334 when such microbial consortia were employed [Munawar et al., 2007]. These local isolates may 335 potentially be utilized in various bioleaching processes of low or high-grade ores. The potentials 336 of these acidophilic extremophiles may also be utilized for recycling the local electronic wastes. 337 338 3. Potential applications of extremophiles at regional level-Recent trends 339 Since last decade, Pakistan has been facing a serious economic crisis due to war on terror, natural 340 disasters, social and political instabilities and energy crisis. Taking energy crises into account, the 341 current energy need of the country surpasses total generation [Khalil and Zaidi et al., 2014]. The 342 country is mostly relying on hydel power, oil and nuclear power stations for electricity 343 generation. These energy crises have rendered investors reluctant to invest and even driving the 344 established industries to back out. The Pakistan Council of Renewable Energy Technologies 345 (PCRE) was established to overcome the energy crises and to search and propose alternative 346 energy sources; biofuel, for instance. The ministry of petroleum and natural resources of 347 Pakistan, on the other hand, is the pioneer in the country that introduced biogas plants. From 348 1974 to 1987 the ministry installed 4137 biogas plants in different cities [Mirza et al, 2008; Bond 349 and Templeton, 2011]. Later, in 2001 the PCRE technologies built 1200 more biogas plants 350 [Bond and Templeton, 2011]. It is reported that a total of 5357 biogas plants are operational in 351 the country [Bond and Templeton, 2011]. Most of these biogas plants harbor complex microbial 352 communities, where the microbes of interest would have to compete for nutrients with other 353 dominating groups of microorganisms which eventually limit the biogas production. Therefore, 354 use of enriched pure cultures of biogas producing microorganisms would be of great interest in 355 enhancing the yield of biogas plants. Despite the fact that the maintenance of pure cultures of 356 these microbes would not be possible—as the substrate harbors a number of other species as 357 well—still the higher number of the desired microbes will lead to comparatively efficient 358 production of the biogas. This process would be further benefited if the continuous addition of 359 pure cultures is maintained at various time intervals. To improve the productivity of the biogas 360 plants, there is a need to shift from conventional methods towards recently used advanced 361 approaches. In this instance, the huge number of already implemented biogas plants can be 362 further profited with the use of extremophiles [Qurat-ul-Ain and Qazi, 2014]. Thermophilic 363 processes have been correlated with better production of biogas [Angelidaki and Ellegaard L, 364 2003]. There is much likelihood that utilization of pure cultures of thermophilic microorganisms 365 will enhance the efficiency of the biogas plants. Some of the local extremophiles have been 366 reported capable of producing methane gas (Table 2), they can potentially be employed in biogas 367 plants. The sugar mills industry of Pakistan stands as another sector, where utilization of such 368 processes has great potentials for methane gas production through bagasse degradation. These 369 sugar mills utilize 4670000 tons of sugarcane, leaving an average 1401000 tons of bagasse 370 [Rehman MS, 2013], which can potentially be used to generate 2000 MW of electric power 371 [Mirza et al., 2008]. Meanwhile, only two out of 78 sugar mills of the country, namely the Shakar 372 Ganj Sugar Mills (Jhang) and Habib Sugar Mills (Nawabshah), have adopted such processes for 373 the production of biofuel. Both the mills generate sufficient electricity by growing biogas 374 producing microbes on bagasse, that fulfills not only the energy needs of the sugar mills but even

375 they sell the surplus electricity to the national grid [Malik, 2014]. It is being expected from both 376 the government and the private sector to utilize biotechnological approaches to achieve the 377 country’s energy demands. The Pakistan sugar mills association should convince the fellow 378 industrialists to implant such technologies that are cost-effective and environment-friendly. 379 380 Beside biofuel applications, microorganisms synthesize valuable enzymes that are 381 routinely used in industries like textile, leather, hosiery, and denim. The enzymes of 382 extremophiles are resistant to the extreme processing conditions of industry. To meet the 383 industrial demands of the country, around 500 tons of cellulase and 5000-7000 tons of α-amylase 384 are imported each year costing over $10 million every year on the import of dextransucrase, α- 385 amylase, and protease. These enzymes could be locally produced which in turn can reduce the 386 expenses of industry and generate more jobs [Dawn News, 2009]. This can be achieved by 387 tapping the potential of producing cheap agriculture commodities such as biotechnologically 388 potent enzymes from local bacterial isolates utilizing agricultural wastes (tables 1– 4). Keeping 389 the industrial demand for enzymes, the country is investing in enzyme research sector. The trend 390 for a search of beneficial microorganisms capable of producing biotechnologically potent 391 enzymes has been on the rise. Various, enzyme-research oriented projects are currently running 392 in most of the biotechnological institutes of the country. Recently, an enzyme has been isolated 393 from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with promiscuous 394 pullulanase and α-amylase activities. Additionally, the enzyme was stable at higher temperatures 395 (95-100°C) and a broad range of pH (3.0-8.5). The enzyme has been patented (US 20140227744 396 A1) and the researchers are interested in its bulk production. National Institute of Biotechnology 397 and Genetic Engineering (NIBGE), being one of the leading biotechnological research institutes 398 of the country, is currently focusing on extremophiles as a source of novel and biotechnologically 399 potent enzymes [Ghaffar et al., 2011]. Researchers from industrial biotechnology group at 400 NIBGE have isolated several other enzymes that have promising applications in pulp, bio-stone 401 washing and bio-finishing of garments. Several fiber-degrading enzymes are in the process of 402 commercialization to be applied in the poultry and ruminant feed industries (personal 403 communications and NIBGE website). Additionally, NIBGE has taken steps forward for the 404 qualitative and quantitative improvement of enzymes, utilizing various approaches, such as 405 mutagenesis [Arshad et al., 2014] and solid state fermentation [Tabassum et al., 2014]. Similarly, 406 Dr. Abdul Qadeer Khan Institute of Biotechnology and Genetic Engineering (KIBGE) of Karachi 407 University recently announced the mass scale production and commercialization of five 408 industrially important enzymes, including dextransucrase, α-amylase and protease [Dawn News, 409 2009]. Several other universities and research centers of the country are also trying to optimize 410 conditions for mass level production of various enzymes. However, NIBGE and KIBGE are 411 currently the leading institutes in enzyme research in Pakistan. 412 413 4. Conclusions and perspectives 414 Pakistani extreme environments, being diverse, unique and rich in terms of abiotic and biotic 415 components home a variety of extremophiles. The conventional based approaches utilized so far 416 to study the microbial diversity of these extreme environments are not sufficient lacking even a 417 single report on the structural based diversity of any of the extreme environments of the country. 418 Moreover, some of the extremophilic isolates from these environments are not true extremophiles 419 because extremotolerants are usually confused with extremophiles by the local graduates. 420 Additionally, some of the extreme environments of the country have been completely disregarded 421 so far (e.g. Peatlands). There are numerous peatlands in northern areas of Pakistan, such as

422 Deosai peatland, located in Skardu, Gilgit-Baltistan (Latitude: 35° 03' 40" N and Longitude: 75° 423 28' 34" E). This peatland is situated at an average elevation of 13,497 feet (4,114 M) above the 424 sea level, which makes it one of the highest plateaus in the world. Other unexplored extreme 425 environments include the enormous number of caves in Pakistan. To date, there are more than 426 100 recorded caves in Pakistan [Gunn, 2004]. Few examples of caves from Pakistan include “Pir 427 Ghaib Gharr Gharra” Balochistan (1,275 M), “Murghaghull Gharra” Balochistan (576 M) and 428 Kach Gharra (Juniper shaft Cave) Balochistan (131 M). Microbial diversity is considered as a 429 function of the environment, therefore these unexplored environments might harbor 430 biotechnologically potent microbes and unique diversity. Exploration of extremophiles with 431 modified culture conditions and structural or functional based metagenomic approaches may lead 432 to the isolation of novel microbes and may explore their biotechnological potentials [Vester et al., 433 2015]. The above-narrated strategies will help to isolate new extremophiles that may encode 434 industrially valuable enzymes. Most of the microorganisms in nature are not culturable, and one 435 might imagine the diversity and presence of novel genes for various biotechnologically potent 436 enzymes from the majority of unculturable microorganisms. Function-driven metagenomics is an 437 effective way to search for novel genes and enzymes from the diverse microorganisms. Such 438 biotechnological approaches may help to domesticize extremophiles from their inhabitable 439 natural locations and the efforts may bring oversized impact in our lives. 440 441 5. Conflict of interest 442 The authors declared no conflict of interest 443 444 6. Author Contributions 445 RK, MK and AZ. Through the idea and conceived and organized the manuscript. RK, MK, AZ, 446 IK, YC, NR, SA, RU and ZB interpreted and analyzed the data and prepared the manuscript. RK, 447 MY, and JQ, critically evaluated the manuscript. All of the authors read and approved the final 448 version of the manuscript before submission. 449 450 7. Funding 451 Not applicable 452 453 8. List of abbreviations 454 PCRE: Pakistan Council of Renewable Energy Technologies 455 NIBGE: National Institute of Biotechnology and Genetic Engineering 456 KIBGE: Dr. Abdul Qadeer Khan Institute of Biotechnology and Genetic Engineering 457 458 9. Acknowledgments 459 We thank Dr. Junaid Khaliq for critically reading this paper. 460 461 10. References 462 Abbas, S., Ahmed, I., Kudo, T., Iida, T., Ali, G. M., Fujiwara, T., Ohkuma, M. (2014). Heavy 463 metal-tolerant and psychrotolerant bacterium Acinetobacter pakistanensis sp. nov. isolated from a 464 textile dyeing wastewater treatment pond. Pak. J. Agric. Sci. 51, 595–608.

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739 Figure Legends

740 Fig. 1 Map of Pakistan showing different localities from which extremophillic bacteria were 741 isolated. The sample source area names are indicated with closed red circles.

742 Fig. 2 Map of Pakistan showing various natural extreme habitats of extremophiles in Pakistan, 743 marked with closed numbered red circles. 1, 4; Salt mines of salt range, 2; various glaciers, 3; hot 744 springs and 5; desert areas.

745 746 747

748 749 Table 1 Thermophiles, isolated from various environments of Pakistan 750 Organism(s) Source Source Biotechnological potential References location S. Thermosulfidooxidans sp. Uranium mines DG Khan Bioleaching of metal ions [Ilyas et al., 2007] B. cereus sp. Soil Lahore Antimicrobials production [Javed et al., 2009] T. aquaticus strains Hot spring Gilgit Hydrolases and Taq polymerase [Javed et al., 2012] production G. pallidus sp. Hot spring Tatta Pani Gelatin hydrolase production [Zahoor et al., 2012] B. licheniformis sp. Soil Faisalabad Thermostable extracellular α– [Niaz et al., 2010] amylase B. cereus sp. Sugar mill Lahore Biofuel production [Chaudhary and Qazi, 2008] Bacillus CTS strain Cellulosic material Lahore Cellulase production [Zaidi, 2007] S. thermosulfidooxidans Cement plant Nizampur Biomining potential [Munawar et al., 2007] and A. caldus strains B. licheniformis (PB1) Desert Sindh Thermostable protease [Ghumro et al., 2011] Bacillus sp. PCSIR EA–3 Air Rawalpindi Thermostable protease [Qadar et al., 2009] Bacillus sp. CEMB10370 Detergent factory Lahore Thermostable alkaline protease [Khan et al., 2011] Bacillus strain SAT–4 Desert Thar Sindh Source of antimicrobials [Muhammad et al., 2009] Bacillus sp. WA21 Hot spring Karachi Biopolymer degradation [Asad et al., 2011] Bacillus sp. GQ 301542 Hot spring Tatta Glucanase and Cellulase production [Rafique et al., 2010] G. thermopakistaniensis Hot spring Northern Production of complex sugar [Siddiqui et al., 2014] Areas hydrolyzing enzymes 751

752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774

775 Table 2 Acidophiles and other extremophiles isolated from various environments of Pakistan 776

Organism(s) Source Organism Source Biotechnological Reference nature location potential

L. bulgaricus, L. casei, L. Yogurt Acidophile Lahore Probiotics and Dairy [Aslam and Qazi, 2010] acidophilus and L. salivarius industry Bacillus strain Tannery waste Acidophile Lipase production [Ghori et al., 2011] Bacillus strain (RM16) Hot spring Acidophile Karachi Thermostable α– [Hassan et al., 2011] amylase L. brevis, L. plantarum and Bakeries point Acidophile Faisalabad Fermented bakery [Saeed et al., 2009] L. fermentum products L. Paracasei Curd Acidophile Lahore Dairy industry [Ikramulhaq and Muktar, 2006] Acidithiobacillus Black shale Acidophile Tarbela Starch and gelatin [Khan and Haq, 2012] ferrooxidans hydrolysis T. thiooxidans, T. Water hyacinth Acidophile Taxila Bioleaching of metals [Mehmood et al., 2009] ferrooxidans and Azotobacter S. thermosulfidooxidans Coal heap Acidophile Faisalabad Bioleaching of metals [Ilyas et al., 2007] S. thermophiles and L. Homemade Acidophile Faisalabad Mozzarella cheese [Sameen et al., 2010] bulgaricus yogurt production. Methanosarcina mazei and Biogas plant Methanophile Karachi Methane production [Rajoka et al., 1999; Methanobacterium formicium Siddiqi and Robinson 1993] Various Bacillus and Organic soil Alkalophile Kamalia Leather processing and [Nadeem et al., 2007] Klebsella strains sample detergent industries

Pseudomonas, Arthrobacter, Glaciers Psychrophile Batura, Alkaline phosphatase [Ahmad, 2010] Stenotrophomonas and Hopper production Bacillus generas and Passu glaciers 778

779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795

796 Table 3 Metallophiles isolated from various environments of Pakistan 798 Organism(s) Source Source Biotechnological potential References location Citrobacter freundii, Wastewater plant Sheikhupura Arsenic reductase [Shakoori et al., 2010] K.oxytoca and B. anthracis production sp. Pseudomonas lubricans Metal laden Sheikhupura Heavy metal detoxification [Rehman et al., 2010] industrial potential wastewater Sulfobacillus Uranium rich Khewra Integrase enzymes [Ghauri et al., 2006; Thermosulfidooxidans environments and production and Uranium Ghauri et al., 2003] salt mine leaching Pseudomonas sp. strains Electroporating Gujranwala Chr (VI) detoxification [Sultan and Hasnain, units 2000] Vibrionacea strains Drainage Lahore Environmental restoration [Afrasayab et al., 2002] wastewater of Mercury S. thermosulfidooxidans and Duddar deposit Lasbela Metal bioleaching, [Rehman et al., 2009] A. ferrooxidans biohydormetallurgical potential Bacillus sp. KS21 Industrially Kasur Arsenic reduction [Rehman et al., 2013] contaminated sites Cellulosimicrobium sp. Industrially Kasur Chromium reduction [Rehman et al., 2013] contaminated sites A. pakistanensis Textile dyeing Islamabad Bioremediation [Abbas et al., 2014] wastewater treatment pond

799

800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819

820 821 Table 4 Halophiles isolated from various environments of Pakistan 822 Organism(s) Source Source Biotechnological potential References location Bacillus pakistanensis sp. Salt mine Karak β-galactosidase, gelatin hydrolase and catalase [Roohi et al., 2014] production B. fumarioli Salt mine Khewra Protease production [Akhtar et al., 2008] B. sphaericus Salt mine Khewra Protease production [Akhtar et al., 2008] B. amyloliquefaciens Salt mine Khewra Amylase, CMCase, Xylanase, Cellulase and [Akhtar et al., 2008] Protease production B. cereus Salt mine Khewra Amylase, CMCase, Xylanase, Cellulase and [Akhtar et al., 2008] Protease production S. arlettae Salt mine Khewra Amylase and Protease production [Akhtar et al., 2008] S. gallinarum Salt mine Khewra Amylase production [Akhtar et al., 2008] B.licheniformis KL-176 Salt mine Khewra Amylase, CMCase, Xylanase, Cellulase and [Akhtar et al., 2008] Protease production B. pumilus Salt mine Khewra Amylase, CMCase, Xylanase, Cellulase and [Akhtar et al., 2008] Protease production 21 different halotolerant Salt mine Karak Potential source of salt stress responsive genes [Roohi et al., 2012] strains from various generas Cellulomonas Paddy grains Islamabad Plant growth promotion [Ahmed et al., 2014] pakistanensis Kushneria pakistanensis Salt mine Karak Plant growth promotion [Bangash et al., 2015] 823 Abbreviations: CMCase; Carboxymethyl cellulase 824 825

826

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