Patent Application: US-201615211396-A

Abstract:
a granular or particulate composition of matter that includes algae and bacteria is described . the algal - sludge granules are generated by incubating a wastewater system with algae under specific quiescent conditions with illumination . once the algal - sludge granules are present , it is no longer necessary to maintain quiescent conditions , and reaction with wastewater under stirred conditions is possible . the methods described include ab initio generation of the algal - sludge granules , use of the algal - sludge granules to remediate wastewater , and use of the algal - sludge granules to generate biomass . it is believed that the remediation of wastewater by algal - sludge granules will save the energy for wastewater treatment , recover the energy in wastewater in the form of biomass , and reduce the wastewater treatment carbon footprint .

Description:
a biogranule is a dense , typically , spherical aggregate of microorganisms that can be considered a self - immobilized biofilm formed in the absence of solid substratum . we produced a novel biogranule that results from transformation of activated sludge under a unique incubation condition . the novel biogranule , referred to hereinafter as an algal - sludge granule , is naturally formed ( transformed naturally ) from activated sludge without adding any further biological or chemical agents and composed of cyanobacteria , algae , bacteria , and protozoa within one granular biomass ( see fig1 a - 1b ., fig2 a - 2b ., fig3 a - 3c , fig4 a - 4h , fig5 a - 5c , fig6 , fig7 a - 7d , fig8 a - 8b , fig9 a - 9b , fig1 a - 10b , fig1 a - 11b ). the cohabitation of microalgae ( algae and cyanobacteria ) and bacteria , and even protozoa , within the biogranule enables a consistent , efficient symbiotic wastewater treatment process : bacteria degrade organic matter , using o 2 produced by microalgae ; in turn , microalgae harvests co 2 , produced from organic matter degradation , for photosynthesis . this unique biomass grows in large - size granules , typically 0 . 2 to 10 mm , enabling the algal - sludge granule process to perform at a high volumetric loading rate with excellent biomass separation from water , thus overcoming a major algae process challenge . see fig1 a - 1c . as is demonstrated herein below , we have shown that various source of wastewater sludges provide microorganisms such as algae , cyanobacteria , bacteria and protozoa that are effective in performing the methods of the invention . the invention creates algal - sludge granules from activated sludge by incubation of activated sludge in a quiescent , batch condition illuminated with natural and / or artificial light in either an open or a closed vessel . see fig1 a , fig2 a - 2b , fig3 a - 3c , fig4 a - 4h . for the purposes of the present discussion , the term “ quiescent ”, as applied to a fluid system , will be understood to mean a system in which there is no deliberate mechanical stirring , and no deliberate imposition of thermal , compositional , or density gradients that would lead to convection or other driven fluid flows that occur in a gravitational field , such as the gravitational field of the planet earth . note that once the granule is formed the stirring of incubation will not pose a problem . see below for further details . the activated sludge described in this invention disclosure is referred to mixed liquor , thickened mixed liquor ( also called sewage sludge , returned activated sludge , waste activated sludge ), or biofilms present and used in water and wastewater treatment systems . fig2 a is an image of laboratory incubation of activated sludge to produce algal - sludge granules . activated sludge stored in the vessel could either settle or float and granulation typically proceed from that initial phase of biomass . fig2 b and fig3 a - 3c are images showing the progression in biogranulation of settled activated sludge ( from left to right ). fig4 a - 4h are images showing the progression in biogranulation of floating activated sludge . the invention also involves using any photochemical treatment of activated sludge , either with or without the addition of external source of microalgae ( cyanobacteria and / or green algae ), to make biogranules that contain substantial numbers of microalgae ( cyanobacteria and / or green algae ), bacteria , and protozoa within the same granular biomass . the novel algal - sludge granules formed from activated sludge are spherical bioaggregates , or sometimes disc - like biomass , populated with substantial numbers of microalgae ( cyanobacteria and / or green algae ), bacteria , and protozoa . fig6 depicts a typical structural composition of algal - sludge granule formed from activated sludge by our incubation method . the thick outer layer is mostly composed of motile filamentous cyanobacteria . the inner layer usually has more green algae and bacteria , with some filamentous cyanobacteria . the center of the granule is primarily composed of sludge - like matter . fig8 a and fig8 b are scanning electronic microscopic ( sem ) images of a whole and cross - sectioned granule , revealing the detailed architecture of the granule and its associated microbial composition . the settled or floating granule is not substantially different with respect to their structural and microbial composition . the original activated sludge used to generate algal - sludge granules was typical activated sludge collected from the aeration basin at a local wastewater treatment plant ( amherst , mass .) and did not contain measurable concentration of chlorophyll . only microscopic analysis could inform that a very small number of algae and cyanobacteria or their cysts or spore - like materials were present within the sludge flocs . this indicates that the photochemical reaction induced substantial growth of these native cyanobacteria and algae within sludge flocs and their growth takes place along forming a unique shape of biomass , algal - sludge granules . we conducted control experiments by incubating activated sludges under dark conditions . we did not observe the growth of algae within the stored activated sludge ( thus , biomass did not change to green color ) and the sludge also did not granulate . therefore , we conclude that illumination is a driver of the process , which appears to include processes such as photosynthesis . our studies have shown that substantial growth of filamentous cyanobacteria , especially motile filamentous cyanobacteria , such as genus oscillatoria , phormidium , and microcoleus , within sludge plays a key role in forming an overall structure of the granule and transforming sludge flocs to algal - sludge granules . the growth of motile filamentous cyanobacteria in high - cell density in sludge is very important for granulation because their gliding motility leads to the formation of interwoven structure of cyanobacterial mat , which in our granule encompasses inner region of biomass , forming the granular structure . small addition of edta into sludge completely inhibited granulation ( fig1 a ) while small addition of ca 2 + significantly enhanced granulation ( fig1 b ). since extracellular proteins that are involved in cell motility of many filamentous cyanobacteria are ca 2 + - dependent proteins , these results indicated that substantial growth of filamentous cyanobacteria and their motility have an important role in the formation of algal - sludge granules from activated sludge . the novel biogranules also contain large amounts of extracellular polymeric substances ( eps ) supporting granulation . significant fractions of these eps are slimes produced by filamentous cyanobacteria , which are essential for gliding motility of many filamentous cyanobacteria . detection of covered slime and empty slime tubes inside the granule support this notion . see fig1 a and fig1 b . we learned that eps of original activated sludge is also important for granulation . dissociation or stripping of eps from activated sludge before incubation significantly retarded or inhibited granulation . this also indicates why flocculent nature of sludge is important for transforming activated sludge into algal - sludge granule . the first discovery of formation of algal - sludge granules was made through incubation of activated sludge in a 20 ml scintillation vial sitting next to lab windows ( under natural light conditions ) for several months . later , we incubated activated sludge in scintillation vials under artificial light conditions and also found that algal - sludge granules were generated from activated sludge . we believe that the reason this algal - sludge granule forms in our system , but does not form in conventional water / wastewater treatment plants , is that in the incubation system we used , there is essentially no stirring of the solution . by comparison , conventional water / wastewater treatment plants deliberately pump , stir and / or aerate the water , wastewater , and / or sludge , thereby making a quiescent system effectively impossible . we believe that stirring of the system also does not promote the formation of an interwoven mat of motile filamentous cyanobacteria ; thus , no effective granulation . we have discovered that once the algal - sludge granules are formed and present , stirring of the system does not pose a problem , because the algal - sludge granules are already established . the original generation of the initial algal - sludge granules , thus , requires quiescent incubation conditions . from 2011 , we have conducted many different sets of incubation experiments using activated sludge collected from six different wastewater treatment plants , and every time we confirmed granulation of activated sludge into algal - sludge granules . our lab notebooks and electronic files include all detailed information regarding the observation about the experiments , such as photos , drawings and descriptions about the granules . the dates of each experiment set started are listed as follows : in 2011 : october 4 , november 22 , december 1 in 2012 : february 23 , april 21 , november 12 in 2013 : january 28 , april 2 , june 20 , november 13 we reduced our invention to practice during our research period from october 2011 to june 2013 , during which time we did not receive any federal funds in support of this invention . the invention is also using the original algal - sludge granules generated by the methods described above as seed or inoculum for the algal - sludge granule process in batch or any flow - through reactors . the algal - sludge granule process is mainly used for wastewater treatment , nutrient removal , recovery of resources ( such as nutrients or scarce elements ), production of high - value byproducts of microalgae , and / or bioenergy feedstock generation but is not limited to these purposes . the invention is also inoculating and / or seeding offspring algal - sludge granules in the new bioreactor for the algal - sludge granule process . this novel algal - sludge granule process could utilize natural light and / or light from the artificial lighting device to promote photosynthesis of algal - sludge granules . the process can happen under 24 - hr light condition or periodic light condition by adjusting light provision . for the purpose of achieving nitrogen removal by nitrification and denitirification , the process can go under light / dark reaction , even during the day time . also , to support the balance in the growth of microalgae and bacteria in the granule , the light condition could be adjusted . some examples of alternating light condition of the algal - sludge granule process are shown in fig1 , fig1 and fig1 . fig1 is a schematic of algal - sludge granule process for wastewater treatment and nutrient removal under natural light conditions . fig1 is a schematic of algal - sludge granule process for wastewater treatment and nutrient removal under alternating light and dark conditions . dark conditions can be maintained by covering part of the reactor . light conditions can be adjusted by exposing the reactor to either sunlight or artificial light . fig1 is a schematic of photo - bioreactors that adopt algal - sludge granule process for wastewater treatment , nutrient removal and / or bioenergy feedstock generation . light conditions can be different based on desired operation conditions . the algal - sludge granule process can work with or without the addition of external source of co 2 . the addition of co 2 during the algal - sludge granule process is expected to generate more biogranular biomass , improving the yield of bioenergy feedstock . the algal - sludge granule process can be used for the main stream , side stream ( for high - strength wastewater , such as anaerobic digestion liquor ), or effluent - polishing treatment for wastewater and nutrient treatment at wastewater treatment plants . since algal - sludge granules are large and dense , they are easily separated from treated water and wastewater , which will enable a simple biomass separation strategy , permitting a very small bioreactor and settling tank for the wastewater and nutrient treatment process . furthermore , ultimate harvesting of algal - sludge biomass will be accelerated . we have operated the algal - sludge granule process in sequencing batch reactors ( sbrs ) and found that the process can be sustained in this bioreactor application . fig1 a and fig1 b are images of the reactor set up and operation of sbr tested in the laboratory . fig1 shows an image of algal - sludge granules growing in sbr treating real wastewater collected from the local wastewater treatment plant ( amherst , mass .) fig1 a and fig1 b show the data obtained from the reactor operation of sbr fed real wastewater collected from the local wastewater treatment plant ( amherst , mass .). the invention can also be used to retrieve bioenergy from harvested algal - sludge granules by conducting physical , chemical , or biological treatment , including anaerobic digestion , of biogranules . we believe that the biomass of algal - sludge granule itself is novel . finding that activated sludge can be processed to create algal - sludge granules is a new discovery . as a consequence , we believe that the methods that create algal - sludge granules , which are described above , are novel . similarly , the bioprocesses that incorporate and apply these novel algal - sludge granules for wastewater treatment , nutrient treatment , bioenergy feedstock generation , and / or other purposes are also believed to be novel . algal - sludge granules generate o 2 by themselves due to the symbiotic oxygenation from photosynthesis within the granule . the evidence of in - situ o 2 generation can be seen from the data shown in fig1 and fig1 . fig1 a is a graph of temporal changes in the flux of o 2 into and out of a granule . fig1 b is an image showing a microelectrode that measures o 2 at two positions about 5 μm apart within the liquid boundary layer on the exterior of an algal - sludge granule . for the data shown in fig1 a , we measured the flux of o 2 very near the granule &# 39 ; s surface . for flux measurement we used non - invasive microtest technology ( nmt ). as the data show , when the algal - sludge granule was placed in the new wastewater media , the o 2 flux was negative , indicating o 2 transport from solution into the granule . when light was provided , the measured net o 2 flux was directed outward from the granule , indicating that o 2 produced by photosynthesis diffused out toward the bulk liquid . later , when the light was turned off , the flux of o 2 reversed immediately with the bulk o 2 diffusing into the granule because of continuous organic matter degradation inside the granule by sludge biomass . fig1 is a graph of deoxygenation and reoxygenation in the bottle containing algal - sludge biogranules in response to the absence and the presence of light . we also conducted bulk dissolved oxygen ( do ) measurements in a headspace - free , closed do bottle using biomass harvested from the sbr algal - sludge granule system ( fig1 ). the bulk do in the reactor during the light period was supersaturated at approximately 12 mg / l . when the do bottle containing granules was covered with aluminum foil ( i . e ., dark condition ) the do was consumed rapidly , indicating that photosynthesis was turned off and o 2 consumption by internal sludge biomass prevailed . when the same bottle was re - exposed to light there was immediate do regeneration that reached 18 mg / l in the bottle ( again , this was a closed do bottle ), indicating that photosynthesis was immediately initiated as the light was available and the granule pumped o2 into the bulk liquid , which implied that do within the granule was also saturated . we believe that bacteria and protozoa that cohabitate in the algal - sludge granule utilize o 2 , produced internally by symbiotic microalgae , for organic matter removal and nutrient treatment . thus , the algal - sludge granule process eliminates or significantly reduces the need of external aeration to dissolve o 2 into wastewater , which currently causes the highest energy demand at wastewater treatment plants . because of large size and high density , algal - sludge granules readily separate out from water , enabling a simple biomass separation strategy ( small bioreactor and settler ) and control of algal process , which are the biggest challenges in algal processes or algae - based wastewater treatment . in addition , symbiosis of algae and bacteria within the same granular biomass facilitates the engineering of an algae process for wastewater and nutrient treatment . thus , algal - sludge granules and the bioprocesses that adopt these novel biogranules have great potential to achieve three important and timely outcomes , which are to : 1 ) treat wastewater and nutrients with minimal energy investment , 2 ) reduce the wastewater treatment carbon footprint , and 3 ) recover chemical energy laden in wastewater in the form of biofeedstock . we are convinced that our algal - sludge granule is a novel biogranule , which also enables us to treat wastewater and nutrients and retrieve the chemical energy laden in wastewater in novel ways . the novel algal - 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( 1995 ) envelope structure of four gliding filamentous cyanobacteria . journal of bacteriology 177 , 2387 - 2395 . although the theoretical description given herein is thought to be correct , the operation of the devices described and claimed herein does not depend upon the accuracy or validity of the theoretical description . that is , later theoretical developments that may explain the observed results on a basis different from the theory presented herein will not detract from the inventions described herein . any patent , patent application , patent application publication , journal article , book , published paper , or other publicly available material identified in the specification is hereby incorporated by reference herein in its entirety . any material , or portion thereof , that is said to be incorporated by reference herein , but which conflicts with existing definitions , statements , or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material . in the event of a conflict , the conflict is to be resolved in favor of the present disclosure as the preferred disclosure . while the present invention has been particularly shown and described with reference to the preferred mode as illustrated in the drawing , it will be understood by one skilled in the art that various changes in detail may be affected therein without departing from the spirit and scope of the invention as defined by the claims .