The Col­ors of the Mi­cro­bial Rain­bow

by Gemma Reguera

Our ap­pre­ci­a­tion of the col­ors of na­ture is lim­ited by the nar­row wave­lengths of the elec­tro­mag­netic spec­trum that our eyes can de­tect. This por­tion of the elec­tro­mag­netic spec­trum be­tween 390 and 750 nm is what we re­fer to as the vis­i­ble (vis) light or, sim­ply, light.  We can­not see be­low (ul­tra­vi­o­let light) or above (in­frared light) these wave­lengths. Yet this nar­row mar­gin of de­tec­tion al­lows us to see all the col­ors of the rain­bow, spread­ing across the vis­i­ble spec­trum from vi­o­let (short­est wave­length) to red (longest wave­length). You can see the color palette of vis­i­ble light in the rain­bow up in the sky on a rainy day, as light is dis­persed by wa­ter droplets. You can also re­veal the full color of the vis­i­ble spec­trum us­ing a glass prism: the change of speed of the light as it crosses the glass medium changes the di­rec­tion of the light waves and en­ables their dis­per­sion.

The only part of the elec­tro­mag­netic spec­trum that is vis­i­ble to the hu­man eye is in the nar­row re­gion be­tween 390 and 750 nm, which con­tains all the col­ors of the rain­bow and is re­ferred to as the vis­i­ble or vis light spec­trum (ver­ti­cal ar­row at the bot­tom). Source

Some nat­ural sur­faces can pro­duce even more com­plex op­ti­cal ef­fects than rain droplets or a glass prism by se­lec­tively re­flect­ing light of spe­cific wave­lengths and, there­fore, spe­cific col­ors. One op­ti­cal ef­fect, in par­tic­u­lar, the one called iri­des­cence, pro­duces some of the most in­tense col­orations in na­ture such as the rain­bow-like col­oration of soap bub­bles, the in­side of some shells, and the bright col­ors of the ex­oskele­ton of some in­sects. The word iri­des­cence orig­i­nates from the Greek iris, which means 'rain­bow', and refers to the op­ti­cal prop­erty of some sur­faces to change color and its in­ten­sity with the il­lu­mi­na­tion or the view­ing an­gle. Iri­des­cent sur­faces are uniquely struc­tured in a way that causes the re­flected light waves to in­ter­act phys­i­cally with each other. The crests and troughs of the re­flected light waves some­times align (they are 'in phase') and re­in­force each other, thus in­creas­ing the in­ten­sity of the re­flected color. By con­trast, if the re­flected light waves are out of phase, they can can­cel each other out and those par­tic­u­lar col­ors never man­i­fest. The fi­nal ef­fect of this op­ti­cal in­ter­fer­ence is the pro­duc­tion of one or more pre­dom­i­nant col­ors, the type and in­ten­sity chang­ing with the an­gle of il­lu­mi­na­tion and/or ob­ser­va­tion. Thus, iri­des­cent col­ors are 'struc­tural': they do not re­sult from pig­men­ta­tion but from phys­i­cal in­ter­ac­tions be­tween light and sur­faces.

Iri­des­cence from soap bub­bles (left), shells (mid­dle) and the ex­oskele­ton of a golden stag bee­tle (right). Source

Al­though iri­des­cence is a wide­spread phe­nom­e­non in na­ture, I was gen­uinely sur­prised to read a re­cent pa­per by Eric Rosenfeld's group pub­lished in Ap­plied and En­vi­ron­men­tal Mi­cro­bi­ol­ogy re­port­ing that sev­eral bac­te­ria, in­clud­ing some well-known lab­o­ra­tory strains of Pseudomonas aerug­i­nosa and Haemophilus in­fluen­zae, are iri­des­cent. The pa­per be­gins with a  fine in­tro­duc­tion about what is known in the field. Bac­te­r­ial iri­des­cence was first re­ported in 1904 but has been loosely and poorly de­scribed there­after. Many re­ports used (or mis­used) ep­i­thets such as "shine," "sheen," "glis­ten­ing", "metal­lic ef­fect," "bright", "lus­ter," "glow," "glis­ten," or "rain­bow-like" For ex­am­ple, the au­thors showed that the 'metal­lic iri­des­cence' pre­vi­ously re­ported for some strains of P. aerug­i­nosa is not an­gle-de­pen­dent. Thus, these colonies are not truly iri­des­cent. In some cases, flu­o­res­cence was mis­taken as iri­des­cence. I must ad­mit that I have used some of these terms loosely in the past, bliss­fully ig­no­rant of what I was observing.The re­searchers uni­fied these ep­i­thets em­ploy­ing a rig­or­ous clas­si­fi­ca­tion of bac­te­r­ial iri­des­cence by us­ing two mi­cro­scopic tech­niques: epi-il­lu­mi­na­tion (where il­lu­mi­na­tion and de­tec­tion take place on the same side of the sam­ple) and trans-il­lu­mi­na­tion (which de­tects the light trans­mit­ted through the sam­ple). They in­ves­ti­gated and de­scribed in de­tail the iri­des­cent prop­er­ties of colonies of sev­eral strains, in­clud­ing an iri­des­cent strain (strain BK) of the ma­rine bac­terium Cel­lu­lophaga lyt­ica (for­merly known as Cy­tophaga lyt­ica). This strain was iso­lated from the sur­face of a red anemone and grew into colonies dis­play­ing a glit­ter-like green col­oration un­der di­rect epi-il­lu­mi­na­tion. Other strains of C. lyt­ica avail­able in pure cul­ture, in­clud­ing the only se­quenced strain of the group (DSM7489), were ei­ther non-iri­des­cent or ex­hib­ited low-in­ten­sity iri­des­cence. In fact, the in­tense green iri­des­cence dis­played by C. lyt­ica strain BK is de­scribed by the au­thors as 'un­matched in the bac­te­r­ial king­dom' and to ri­val that ob­served in some in­sects and ver­te­brates.

Some ex­am­ples of bac­te­r­ial iri­des­cence in lin­ear streaks of colonies ex­am­ined with epi- and trans-il­lu­mi­na­tion. The pre­dom­i­nant col­oration of the colony streak was used to de­fine iri­des­cence cat­e­gories: rain­bow (dif­fuse (D) or edge ®, de­pend­ing on the lo­ca­tion of the rain­bow col­ors along the colony or on the edge); metal­lic (sil­very lus­ter (M)); and glit­ter-like (mid green and red and vi­o­let at the edges of the colony (G)). Source

In­ter­est­ingly, the iri­des­cent col­oration of the C. lyt­ica iso­late was re­spon­sive to the growth medium. Lin­ear colony streaks grown on rich me­dia are yel­low to the naked eye but dis­play glit­ter-like green col­oration in the mid­dle and red and vi­o­let on the colony edges un­der epi-il­lu­mi­na­tion. The glit­ter-like col­ors are clearly iri­des­cent, as they change in type and in­ten­sity with the an­gle of il­lu­mi­na­tion. When grown in a low nu­tri­ent medium (medium LN), the colonies are translu­cent, thus void of any pig­men­tary color, and dis­play the bright green iri­des­cence when il­lu­mi­nated. You can ac­tu­ally watch a movie show­ing the dy­namic changes of the iri­des­cent green col­oration in the translu­cent colony as the an­gle of il­lu­mi­na­tion changes. In a fol­low-up pa­per, the re­searchers pro­vide ev­i­dence link­ing iri­des­cence to cold tem­per­a­ture, salin­ity and con­di­tions of hy­dric stress (low mois­ture), which are all fac­tors these bac­te­ria en­counter in their nat­ural coastal shore en­vi­ron­ment. I can­not help but to men­tally vi­su­al­ize the strik­ing con­trast of the green iri­des­cence of this bac­terium against the red anemone from which it was orig­i­nally iso­lated. Anemones use their col­ors to at­tract fish, ei­ther to eat them or to es­tab­lish mu­tu­al­is­tic re­la­tion­ships (re­mem­ber the movie 'Find­ing Nemo'? Nemo and his dad are clown­fish liv­ing in­side an anemone!). The col­oration also helps de­ter preda­tors. The an­gle-de­pen­dence of the bac­te­ria iri­des­cent col­ors may en­hance the col­oration ef­fects of the anemone and af­fect the be­hav­ior of preys and preda­tors. The anemone, on the other hand, pro­vides the bac­te­ria with a rich nu­tri­ent en­vi­ron­ment, sim­i­lar to the mu­tu­al­is­tic re­la­tion­ship be­tween the clown­fish and anemones.

Lin­ear streaks (ap­prox­i­mately 5‑cm long) of C. lyt­ica strain BK grown on ma­rine agar (a type of rich medium) dis­play green iri­des­cence in the mid­dle and red and vi­o­let in the edges. The type and in­ten­sity of the col­oration de­pends on the an­gle of il­lu­mi­na­tion (22.5° to 135°, from top to bot­tom).

Be­sides its eco­log­i­cal role, it is im­por­tant to un­der­stand what bi­o­log­i­cal mech­a­nisms con­trol bac­te­r­ial iri­des­cence. Nat­ural iri­des­cent sur­faces are highly or­dered struc­tures com­posed of very thin films or lay­ers. The iri­des­cence prop­er­ties of the ex­oskele­ton of some bee­tles re­sult, for ex­am­ple, from the pe­ri­odic arrange­ment of lay­ers of the exoskeleton's struc­tural poly­mer, chitin, within air spaces. In some cases, iri­des­cent sur­faces are com­posed of pe­ri­od­i­cally or­ga­nized nanos­truc­tures, whose arrange­ment re­flects part of the in­ci­dent light at dif­fer­ent an­gles. The ma­te­r­ial of pea­cock feath­ers, for ex­am­ple, is a two-di­men­sional lat­tice of pe­ri­od­i­cally or­dered melanin rods within air spaces. The feather ma­te­r­ial is ac­tu­ally col­or­less and al­most trans­par­ent, but the in­ter­ac­tion of light waves as they are re­flected by the melanin lat­tice pro­duces struc­tural col­ors and gives the feath­ers their bright col­oration. Chang­ing the spac­ing of the rods in the lat­tice af­fects the re­flec­tion of the light waves and their in­ter­ac­tions and changes the fi­nal color that is re­flected from the struc­ture.

Scan­ning force mi­cro­graph of an S‑layer with square lat­tice sym­me­try. The cen­ter-to-cen­ter spac­ing is 13.1 nm. Source

In their pa­per, Rosenfeld's team pro­poses that some mech­a­nism of in­ter­cel­lu­lar com­mu­ni­ca­tion reg­u­lates the pe­ri­odic as­sem­bly of cells within colonies or biofilms so as to pro­duce an op­ti­cally ac­tive cell as­sem­blage that re­flects light as iri­des­cent col­ors. In fact, the re­searchers ob­served a link be­tween cell den­sity and the type of iri­des­cent col­ors of the colony. How­ever, I failed to ap­pre­ci­ate any pe­ri­odic pat­tern of or­ga­ni­za­tion in the mi­cro­graphs they showed of cells within the iri­des­cent colonies. One al­ter­na­tive ex­pla­na­tion could be that the cells pro­duce nanos­truc­tures in pe­ri­odic arrange­ments such that scat­ter light in a co­her­ent man­ner and with tun­able spec­tral char­ac­ter­is­tics. Mi­croor­gan­isms are af­ter all mas­ters of nanoassem­bly. No­tice for ex­am­ple the ex­quis­ite pe­ri­odic arrange­ment of the S layer that en­velops some bac­te­r­ial and ar­chaeal cells and func­tions as struc­tural sup­port, ad­hesin, and per­me­abil­ity bar­rier. The S layer is a sym­met­ric two-di­men­sional lat­tice formed by the pe­ri­odic arrange­ment of a sin­gle pro­tein or gly­co­pro­tein. The type of pro­tein de­ter­mines the thick­ness of the S layer, its pore size and sym­me­try (oblique, square or hexag­o­nal) Sim­i­lar pe­ri­odic nanos­truc­tures could also be re­spon­si­ble for bac­te­r­ial iri­des­cence.

The im­pli­ca­tions of un­der­stand­ing the mech­a­nisms for iridis­cence are far-reached. Al­though there is clearly a light scat­ter­ing ef­fect, the phys­i­cal mech­a­nism be­hind iri­des­cence is still not fully un­der­stood. Ev­i­dence to date sug­gests that sev­eral op­ti­cal phe­nom­ena act to­gether to se­lect for the re­flec­tion of only par­tic­u­lar light wave­lengths, there­fore pro­duc­ing spe­cific col­ors. These stud­ies are dif­fi­cult to carry out in an­i­mals or in­ert sur­faces but could be eas­ier in bac­te­ria. Knowl­edge of how bac­te­r­ial iri­des­cent struc­tures as­sem­ble and the mechanism(s) con­trol­ling their op­ti­cal ef­fects could en­able the syn­the­sis of bio­mimetic ma­te­ri­als with con­trolled op­ti­cal prop­er­ties for the de­vel­op­ment of op­ti­cal coat­ings, paints, cos­met­ics and anti-coun­ter­feit­ing de­vices, just to men­tion a few ap­pli­ca­tions. The field is gen­er­ally known as bio­pho­ton­ics. The fact that bac­te­ria have evolved mech­a­nisms for iri­des­cence could pro­vide new ma­te­ri­als and/or de­signs for the de­vel­op­ment of bio­pho­tonic de­vices at a frac­tion of the cost. It dawned on me that we may have just be­gun to ap­pre­ci­ate the col­ors of the mi­cro­bial rain­bow … and the out­look could not be brighter.

Ref­er­ence

Kientz B, Vuku­sic P, Luke S, Rosen­feld E (2012). Iri­des­cence of a ma­rine bac­terium and clas­si­fi­ca­tion of prokary­otic struc­tural col­ors. Ap­plied and en­vi­ron­men­tal mi­cro­bi­ol­ogy, 78 (7), 2092−2099. PMID 22267664

Gemma is as­sis­tant pro­fes­sor in the De­part­ment of Mi­cro­bi­ol­ogy and Mol­e­c­u­lar Ge­net­ics, Michi­gan State Uni­ver­sity.